Method of improing the growth performance of an animal

ABSTRACT

The invention broadly relates to a method of improving the growth performance of an animal. In particular the present invention relates to a method of improving the growth performance of an animal comprising the step of administering to an animal in need thereof a growth promoting amount of one or more anti-inflammatory agents.

FIELD OF THE INVENTION

The invention broadly relates to a method of improving the growthperformance of an animal. In particular the present invention relates toa method of improving the growth performance of an animal comprising thestep of administering to an animal in need thereof a growth promotingamount of one or more anti-inflammatory agents.

BACKGROUND OF THE INVENTION

In the agricultural industries of many countries, commercial livestockrearing systems have become commonplace. Commercial animal husbandrytechniques have been used in rearing poultry, pigs and cattle and haveresulted in greatly increased production of food products derived fromthese animals.

The commercial raising of livestock requires maximisation of growth rateand feed conversion efficiency so as to reduce the unit cost ofproduction. This requirement has led to the development and widespreaduse of so called “feed additives”.

Feed additives have two general purposes. One purpose is to enhance theperformance of the animal in terms of increased growth rate and/orincreased feed conversion efficiency in healthy and nutritionallyunchallenged animals. The other purpose is to maintain the health of theanimal during periods of trauma or “stress” that inevitably occur in thecurrent practices of intensive rearing thereby keeping the animaldisease free.

In the early 1950's, researchers unexpectedly discovered that anantibiotic ingredient in chicken mash was a “growth factor.” The findingdrastically changed the livestock and poultry industries and was aneconomic boon for pharmaceutical companies. Feed animals are now raisedunder highly controlled conditions and receive specialised feed with avariety of growth promoting additives.

Routine antibiotic administration to animals has become almost universalsince the discovery that the addition of small amounts of antibioticssuch as penicillin, tetracycline and sulfamethasine, to animal feedincreases the growth of pigs and cattle. In 1979, about 70% of the beefcattle and veal, 90% of the swine, and virtually 100% of broilers rearedin the United States consumed antibiotics as part of their daily feed.This use, accounting for nearly 40% of antibiotics sold in the UnitedStates, is estimated to save consumers $3.5 billion a year in foodcosts.

Animals raised under modern conditions optimised for growth promotionreceive rations containing high proportions of protein, usually in theform of soybean or cottonseed meal (meat and bone or blood meal are usedextensively in Australia), and high percentages of grains such as cornor milo, a type of sorghum (wheat and barley in Australia) Feedadditives which have been used include such hormones asdiethyl-stilbesterol, which also increases the rate of weight gain, andtranquillisers (not used widely for pigs) that prevent the effects ofthe stress brought on by confinement conditions from causing disease orweight loss.

Cattle ordinarily require 5 kilograms of feed to produce 1 kilogram ofweight gain. Under optimal growth promoting conditions, and withenriched feed, they gain 1 kilogram with only 3 kilograms of feed.

Although hormones and antibiotics have greatly increased the rate ofgrowth of food animals, the use of such additives has not been withoutproblems. One of the hormones that is commonly used as a growthstimulant, diethyl-stilbesterol or DES, has been shown to be acarcinogen and has been banned from further use in most countries.

When antibiotics are mixed in animal feed, the compounds are spreadthroughout the environment exposing microorganisms to the antibiotics.The constant exposure of the microorganisms to antibiotics putsbiological pressure on the microorganisms to develop a resistance to theantibiotics. This can result in a microorganism that is resistant toantibiotics and causes especially severe and difficult to treatinfections.

An antibiotic-resistant microorganism is potentially a serious pathogenbecause it is difficult to control. If the organism causes an infectionin an animal or in man, the infection may not be controlled withconventional antibiotics. If the infection is serious, there may not betime to determine which antibiotics are effective against the infectingbacteria. The problem has been especially serious when antibioticresistant organisms in meat are consumed by people who themselves takeantibiotics for treatment of disease. Antibiotics inhibit many of thenormal microorganisms in the respiratory and gastrointestinal tracts.This allows the resistant one to proliferate rapidly and produce moreserious disease. The combination of antibiotic resistant organisms fromfood and ineffective antibiotic treatment of people has caused most ofthe deaths due to salmonella food poisoning reported in the UnitedStates in the past several years.

As a result of the increasing appearance of antibiotic resistantbacteria in feed lots and several serious epidemics caused by antibioticresistant bacteria, there is increasing governmental pressure to ban theuse of antibiotics in animal feed. In fact, the World HealthOrganisation and the Australian Government have specified the need touse environmentally friendly alternative methods to control infection.The imminent ban or withdrawal of various antibiotics from livestockfeed and water is likely to (i) increase the incidence of infection inanimals and consequently reduce growth performance (ii) further reducethe health, fertility and breeding performance of animals. Consequently,there is an immediate and increasing need for new, safe and effectivegrowth stimulators of feed animals, as well as a reduction in disease byenhancing health.

Various attempts at promoting animal growth without the use ofantibiotics has been employed many using elaborate and circuitous means.These have included subcutaneous implants of hormones or complex saltshaving cations being made from complexes (see, for example, U.S. Pat.No. 6,197,815; U.S. Pat. No. 3,991,750; U.S. Pat. No. 4,067,994). Noneof these attempts have proven to be simple or effective. Accordingly,there is still a need for a method of improving the growth performanceof animals, which is not reliant on the use of antibiotics or elaboratemethodology.

The applicant has now surprisingly found that the administration ofanti-inflammatory agents, and in particular cytokine receptorantagonists such as interleukin (IL)-1ra, increases the growthperformance of animals while decreasing the amount of antibiotics. Theapplicant also has evidence that a similar growth performance effect canbe achieved by administering soluble cytokine receptors such as TNFαreceptor, IL-6 receptor, IL-4 receptor and IL-8 receptor, or cytokineblocking factors such as TNF blocking factor (Bargetzki et al, CancerResearch 53: 4010-13 (1993); Engelmann et al, Journal of BiologicalChemistry 264: 11974-80 (1989)) or TNF-alpha inhibitor (Engelmann et al,Journal of Biological Chemistry 265: 1531-6 (1990); Seckinger et al,European Journal of Immunology 20: 1167-74 (1990)).

While not wishing to be bound by any particular theory or hypothesis,the applicant considers that the increases in growth performanceobserved in animals that have been administered anti-inflammatory agentsresult from the interplay of four key effects. These are:

1). Anti-inflammatory effect per se;

2). Immunoenhancement effect;

3) Anti-parasitic and anti-microbial effect; and

4). Stress reduction.

Each of these effects, either singly or together, profoundly impact uponthe health and welfare of animals, which in turn affects the growthperformance of animals and thereby the meat quality. For example:

1). Anti-Inflammation

Chronic inflammation is often seen in livestock and relates to immuneactivation triggered by persistent infections and environmental stimuli.Inflammation plays an important role in the initiation of immuneresponses to infection, however, chronic immune activation, particularlyby persistent infection or microbial load, can have deleterious effectson growth and development and can reduce the effectiveness ofvaccination. Consequences of excessive immune activation include theproduction of inflammatory cytokines, fever, inappetence, amino acidresorption from muscle and redirection of nutrients away from meatproduction. Anti-inflammatory agents could reduce the pathology ofchronic immune activation, for example, by reducing the effects ofinflammatory cytokines such as IL-1, IL-6, TGF-β, IL-11, IL-18, IL-12,IL-17, LIF, IFN-γ IL-8, TNF-α and GM-CSF. Alternatively, byadministering soluble cytokine receptors for these inflammatorycytokines ie IL-1 receptor, IL-8 receptor, TNF-α receptor, IL-6 receptoret al, excessive amounts of circulating inflammatory cytokines can bereduced. Cytokine receptor antagonists such as IL-1ra, IL-6ra orTNF-αra, which competitively inhibit the binding of thesepro-inflammatory cytokines to their respective membrane-expressedreceptors, can be used to ameliorate the action of these cytokines.

2). Immunoenhancement Effect

a). TH1/TH2 Immune Responses

The inflammatory response is inextricably tied to the body's immunesystem. Interplay occurs between immune cytokine regulatory networks andthe other regulatory systems of the body. Immune responses to infectionsor antigens can acutely bias each other. The immune response can begeneralised by the type of T cell response. A T helper 1 (TH1) typeresponse is principally involved in cell mediated immunity, whilst a TH2pattern of response is often associated with humoral immunity. TH1 andTH2 type T cell subsets have been implicated in the regulation of manyimmune responses defined by cytokine patterns. TH2 cells express thecytokines interleukin (IL)-4, IL-5, IL-10, and IL-13. IL-3 expression iscommon to both TH1 and TH2 T cells. Whereas, TH1 cells express IL-2,IFNγ, and TNFβ. These TH2 cytokines influence B cell development andaugment humoral responses such as the secretion of antibodies. Bothtypes of TH cells influence each other by the cytokines they secrete.For example, TH2 cytokines, such as IL-10, can suppress TH1 functions.Other cytokines can also influence TH1 or TH2 development such as TNFβ,known to down regulate TH1 responses.

It is known that when there is no invading antigen, the action of Th1cells is considered harmful to the body. Anti-inflammatory agentssuppress the production of IFN-γ in Th1 cells. Also anti-inflammatoryagents suppress the overproduction of Th1 cells and therefore enhancethe production of Th2 (antibody-secreting) cells because these cellscross-regulate one another. This means that by administering particularanti-inflammatory agents the amount of pro-inflammatory cytokines aresuppressed. Alternatively, by administering soluble cytokine receptors,cytokine receptor antagonists, or cytokine inhibitory factors ofcytokines like IL-1, IL-4, IL-8, GM-CSF, IL-6 or TNF-α theoverproduction of cytokines by TH1 cells may be reduced.

b). Antibody Isotype Switching

Antibodies are required to eliminate or protect against infection.Mature B cells undergo the process of switching-antibody class afterantigenic stimulation. TH cells through physical contact and cytokines,referred to as switch factors, regulate isotype switching. Some of thecytokines known to be involved in isotype switching, either alone or incombination, are IL-4, IL-5, TNFβ, IL-1, IL-2, IL-6, and IL-13. IL-4 andIL-5 synergise to enhance IgG1 responses. For example, optimal IgG1responses also requires IL-2. IL-1 can enhance IgA production in thepresence of IL-5. TNFβ induces IgA production.

c). Immune Dysfunction

The genetic potential for most production traits is predetermined bybirth. Many factors (stress, disease, nutrition, immunity etc.)determine whether this potential is achieved. The level and type ofantigen exposure influences and establishes a ‘bias’ of the immunesystem. Most immune responses are biased towards a type that promotesimmunity against bacteria and viruses or a type that promotes immunityagainst many parasites. While the genotype of an animal can influencethis bias, the early experience by the neonate to antigens andinfections can set the immune reactivity towards one or other type. Thisbias is altered depending on subsequent antigen exposure. Breedingprogrammes based on selection for production traits has appeared to beat the expense and detriment of immune competence or reactivity. Thischange has been further exacerbated by the persistent use of antibioticsupplements to water and feed, which has presumably resulted in analtered genetic potential to mount effective-immune responses.

d). Mucosal Immunity

The most prevalent areas of infection in livestock are mucosal sites,primarily the gastro-intestinal tract and the lungs. Thus, the mucosalimmune system is the first line of defence against pathogens anddisease. Cytokines, notably IL-5, IL-4, IL-6 and IL-10, play asignificant role in the regulation and efficacy of immune responses inthe mucosa.

IL-5 and IL-6 act upon B-1 and B-2 subpopulations of lymphocytes in themucosal immune system. Deficiencies in either the production of IL-5 orIL-6, or their receptors result in significantly impaired production ofIgA, the antibody isotype responsible for protective responses in themucosa. Similarly, IL-5, IL-6 and the chemokine MIP-1 alpha have thecapacity to increase IgA responses to mucosal vaccines. IL-4 has animmunoregulatory role in mucosal tissues, primarily by enhancing TH2responses, and thus, enhancing antibody production. IL-4 is consideredessential to the development of mucosal immune responses in the lung,via the involvement of TH2 pathways. Both IL-4 and IL-5 operate inconcert in the lung, with IL-4 committing naive T cells to a TH2phenotype which upon subsequent activation secrete IL-5, resulting ineosinophil accumulation. Furthermore, IL-4 and IL-10 play a role inmucosal tolerance, and thus, help regulate and dampen allergic typeresponses in the gut and reduce the susceptibility of animals to chronicinflammatory conditions of the gut.

3). Anti-Parasitic and Anti-Microbial Effect

a). Anti-Parasitic Effect

Acquired immune responses against pathogens generally fall into one oftwo types, cell mediated (TH1) or antibody mediated (TH2), and this iscontrolled by cytokines. Cytokines involved in TH2 responses areattractive therapeutic targets, as they could protect againstectoparasites and gastrointestinal worms and suppress inflammationinduced by TH1 cytokines. TH2 cytokines induce eosinophilia, IgEsynthesis, and mucus production that enhance protection against wormsand other gut parasites.

b). Anti-Microbial Effects

Microbial infections remain a world-wide problem in terms of economicimpacts and health, despite advances in nutrition, vaccines, chemicalsand antibiotics. The immune response to microbial pathogens incorporatestwo systems of recognition. The first line of defence is innate immunityand this is followed, if required, by the ensuing adaptive response(cell mediated and antibody responses). By decreasing the inflammationwith anti-inflammatory agents including phenylbutazone, flunixinmeglumine and ketoprofen or intravenous DMSO there is an improvement inthe blood flow to the affected tissue, which in turn assists in theinnate immunity to help overcome the infection. This process may beassisted by using vasodilation drugs such as acetylpromazine,phenoxybenzamine, isoxsuprine, pentoxifylline, aspirin and heparin. Analternative approach is to administer the soluble cytokine receptors ofknown inflammatory cytokines such as IL-1, TNF-α, IL-6 and IL-8.

4). Stress Reduction

Many conditions within a commercial environment contribute to areduction in feed intake, growth rate and carcass quality. Despiteextensive research efforts to evaluate the mechanisms by which stressorsaffect performance in many species, the long-standing problems withinthe livestock industries have not been alleviated. Stress, particularlyearly and sustained stress, results in immune dysfunction,Hypothalamic-Pituitary-Adrenalcortical (HPA) activity and an imbalanceof chemicals in the brain. The nervous and immune systems are integratedand form an interdependent neuroimmune network. Depression, physical oremotional stresses activate the endocrine system altering immunologicalfunction, which in turn elicits physiological and chemical changes inthe brain. Likewise, immunological stress in the form of infectionactivates the neuro-endocrine system via cytokines and other solublemediators to induce stress responses which in turn impair productivity.Cytokines mediate interactions between the immune, endocrine and centralnervous systems. Previously believed to be immuno-suppressive, there ismounting evidence that stress induces a shift in TH1/TH2 immuneresponses resulting in immune dysregulation rather thanimmunosuppression. The potential for cytokines to affect homeostaticpathways creates a need to evaluate the activities of the immune system.

SUMMARY OF THE INVENTION

In its broadest aspect the present invention provides a method forimproving the growth performance of an animal comprising the step ofadministering to an animal in need thereof a growth promoting amount ofone or more anti-inflammatory agents.

The anti-inflammatory agents preferably increase or supplements theanimals own anti-inflammatory systems.

The present invention also provides a method for improving the growthperformance of an animal comprising the step of administering to ananimal in need thereof a compound or composition which increases orsupplements endogenous anti-inflammatory agent levels, wherein growthperformance is enhanced relative to the growth performance of an animalwhich has not been administered said compound or composition.

Preferably, the compound or composition is administered prior to,together with, or subsequent to the administration of a growth promotingamount of one or more anti-inflammatory agents.

More preferably the compound or composition comprises antagonists ofpro-inflammatory cytokine receptors. Even more preferably, the compoundor composition comprises antagonists of TNF-α receptor, GM-CSF receptor,IL-6 receptor, IL-1 receptor, IL-4 receptor or IL-8 receptor. Mostpreferably the compound or -composition comprises IL-10,1,8-napthosultam substituted compounds or quinoxaline compounds.

Alternatively, the compound or composition increases the endogenouslevel of anti-inflammatory agents by decreasing the amount ofpro-inflammatory cytokines. Accordingly, the compound or compositioncomprises agents capable of increasing the amount of circulating,soluble cytokine receptors to pro-inflammatory cytokines.

The present invention also provides a method for improving the growthperformance of an animal comprising the step of administering to ananimal in need thereof a composition comprising an anti-inflammatoryagent in conjunction with an antibiotic, optionally in combination witha pharmaceutical carrier, adjuvant or vehicle, wherein said compositionachieves a synergistic growth promoting effect.

Preferably, the anti-inflammatory agent is any soluble cytokinereceptor, cytokine receptor antagonist, cytokine inhibitory factor orbiologically active fragment thereof which has an anti-inflammatoryeffect or an anti-inflammatory agent selected from the group consistingof diclofenac, diflunisal, etodolac, flunix, fenoprofen, floctafenine,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate,mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin,phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic andtolmetin. Preferably, the soluble cytokine receptor or biologicallyactive fragment thereof is selected from the group consisting of TNFαreceptor, IL-6 receptor, IL-1 receptor, IL-4 receptor and IL-8 receptoror a combination thereof that are capable of improving the growthperformance of an animal. More preferably, the soluble cytokine receptoror biologically active fragment thereof is IL-1 receptor.

Preferably, the cytokine receptor antagonist or biologically activefragment thereof is selected from the group consisting of IL-1ra,IL-6ra, IL-8ra and TNF-αra. More preferably, the cytokine receptorantagonist or biologically active fragment thereof is IL-1ra.

Preferably, the cytokine inhibitory factor or biologically activefragment thereof is selected from the group consisting of TNF blockingfactor and TNF-alpha inhibitor.

In one particular embodiment, the anti-inflammatory agents of thepresent invention are formulated into a growth enhancing composition bycombining one or more anti-inflammatory agents together with one or morepharmaceutical carriers, adjuvants or vehicles. More preferably, agrowth enhancing composition is formulated by combining one or moresoluble cytokine receptors, cytokine receptor antagonists, cytokineinhibitory factors or biologically active fragments thereof with eitherone or more other anti-inflammatory agents or pharmaceutical carriers,adjuvants or vehicles. Any known pharmaceutical carrier, adjuvant orvehicle may be used as long as it does not adversely affect the growthpromoting effects of the anti-inflammatory agent(s).

Accordingly, in a second aspect the present invention provides a growthpromoting composition comprising one or more anti-inflammatory agentstogether with one or more pharmaceutical carriers, adjuvants orvehicles. Preferably, the composition comprises anti-inflammatory agentsselected from the group consisting of soluble cytokine receptor,cytokine receptor antagonist, cytokine inhibiting factor or biologicallyactive fragment thereof, diclofenac, diflunisal, etodolac, flunix,fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin,ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone,naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam,tiaprofenic and tolmetin.

More preferable, the composition comprises one or more soluble cytokinereceptor, cytokine receptor antagonist, cytokine inhibitory factor orbiologically active fragments thereof and one or more different solublecytokine receptor, cytokine receptor antagonist, cytokine inhibitoryfactor or biologically active fragments thereof or one or moreanti-inflammatory agent. Most preferably, the composition comprises onesoluble cytokine receptor, cytokine receptor antagonist, cytokineinhibitory factor or biologically active fragment thereof and onedifferent anti-inflammatory agent or a pharmaceutical carrier, adjuvantor vehicle.

Compositions comprising antibiotics assist in limiting the microbialload in an animal, thereby assisting the anti-inflammatory agent toimprove growth performance in the animal. Particularly preferredantibiotics are those already in use in conventional animal productionenvironments. However, in particular, the preferred antibiotic isselected from the group consisting of amoxycylin, ampicillin, apramycin,avoparcin, bacitracin, benethamine, benzathine, ceftiofur, cefuroxime,cephalonium, chlortetracycline, cloxacillin, dimetridazole,erythromycin, kitasamycin, lasalocid, lincomycin, monensin, narasin,neomycin, oleandomycin, oxytetracycline, olaquindox, penicillin,penicillin G, procaine, spectinomycin, streptomycin, tetracycline,tilmicosin, trimethoprim, tylosin, salinomycin, sulfonamides (includingand diaveridine) and virginiamycin or combinations thereof. Mostpreferably, the antibiotic is amoxycylin, lincomycin or spectinomycin.

Depending upon the activity of the anti-inflammatory agent, manner ofadministration, age and body weight of the animal, different doses ofanti-inflammatory agent can be used. Under certain circumstances,however, higher or lower doses may be appropriate. The administration ofthe dose can be carried out both by single administration in the form ofan individual dose unit or else several smaller dose units and also bymultiple administrations of subdivided doses at specific intervals.

It will be understood, however, that the specific dose level for anyparticular animal will depend upon a variety of factors including theactivity of the specific anti-inflammatory agent employed, the age, bodyweight, general health, sex, diet, time of administration, and route ofadministration, rate of excretion and anti-inflammatory agent orantibiotic combination. However, generally the preferred route ofadministration is selected from the group consisting of oral, topicaland parenteral administration.

Parenteral administration includes subcutaneous injections, aerosol,intravenous, intramuscular, intrathecal injection, infusion techniquesor encapsulated cells.

The anti-inflammatory agents or compositions of the invention may alsobe administered as an additive to animal water and/or feed.

The growth performance of an animal may be determined by any knowmeasure including increased growth rate, increased efficiency of feeduse, increased final weight, increased dressed weight or decreased fatcontent. It will be further appreciated by those skilled in the art thatthe improved growth performance of an animal may result fromimmunoenhancement, anti-parasitic or anti-microbial effect,anti-inflammatory effect or stress reduction. More preferably, theimmunoenhancement will result from a TH1/TH2 immune response, antibodyisotype switching, hematopoiesis, improvement in immune function,mucosal immunity, beneficial affects on homeostatic processes such asappetite, endocrine or neural-endocrine processes.

It will be appreciated by those skilled in the art that the methods andcompositions disclosed herein may be useful for any animal for whichimproving the growth performance is a desirable outcome. However, thepresent invention is particularly useful for feed animals ie thoseanimals that are routinely farmed for meat production. Preferably, theanimal is a higher artiodactyl or bird. Artiodactyls include cattle,pigs, sheep, camels, goats and horses. Birds include chickens, turkeys,geese, and ducks. More preferably, the present invention relates toanimals selected from the group consisting of cattle, pigs, sheep,camels, goats, horses and chickens. Most preferably, the animals arecattle, pigs, or sheep.

In a third aspect, the anti-inflammatory agent is administered to ananimal as a nucleic acid molecule encoding said anti-inflammatory agentsuch that upon expression of said nucleic acid molecule in the animal agrowth promoting amount of the anti-inflammatory agent is produced.Thus, the present invention provides a method for improving the growthperformance of an animal comprising the step of administering to ananimal in need thereof a nucleic acid molecule encoding one or moreanti-inflammatory agents, wherein the expression of said nucleic acidmolecule produces an effective growth promoting amount of one or moreanti-inflammatory agents.

The nucleic acid molecule may be DNA, cDNA, RNA, or a hybrid moleculethereof. It will be clearly understood that the term nucleic acidmolecule encompasses a full-length molecule or a biologically activefragment thereof.

Preferably the nucleic acid molecule is a DNA molecule encoding asoluble cytokine receptor, cytokine receptor antagonist, cytokineinhibitory factor or biologically active fragment thereof. Mostpreferably, the DNA encodes a cytokine receptor selected from the groupconsisting of TNFα receptor, IL-6 receptor, IL-1 receptor, IL-4 receptorand IL-8 receptor or a.-combination thereof, or a cytokine receptorantagonist selected from the group consisting of IL-1ra, IL-6ra andTNF-αra.

The nucleic acid molecule may integrate into the animal genome, or mayexist as an extrachromosomal element.

The nucleic acid molecule may be administered by any known method;however, it is preferably injected subcutaneously, intravenously, orintramuscularly or administered as an aerosol.

The amount of nucleic acid that is administered will depend upon theroute and site of administration as well as the particular cytokinereceptor, cytokine receptor antagonist, cytokine inhibitory factor orbiologically active fragment thereof encoded by the nucleic acidmolecule. As described herein, introducing an amount of 200 μg of anucleic acid molecule encoding a cytokine receptor, cytokine receptorantagonist, cytokine inhibitory factor or biologically active fragmentthereof is sufficient to improve growth performance in an animal. Thus,preferably the amount of about 200 μg to 1,000 μg of a nucleic acidmolecule encoding a cytokine receptor, cytokine receptor antagonist,cytokine inhibitory factor or biologically active fragment thereof ispreferably introduced into an animal.

The nucleic acid molecule may also be delivered in a vector such as aporcine adenovirus vector. It may also be delivered as naked DNA.

Accordingly, in fourth aspect, the present invention provides aconstruct for delivering in vivo an effective amount of a cytokinereceptor, cytokine receptor antagonist, cytokine inhibitory factor orbiologically active fragment thereof, comprising:

a) a nucleotide sequence encoding a cytokine receptor, cytokine receptorantagonist, cytokine inhibitory factor or biologically active fragmentthereof;

b) a vector comprising a control sequence wherein the control sequenceis capable of the controlling the expression of the nucleotide sequenceof a) such that a cytokine receptor, cytokine receptor antagonist,cytokine inhibitory factor or biologically active fragment thereof isproduced which in turns improves growth performance in an animal.

Modified and variant forms of the construct may be produced in vitro, bymeans of chemical or enzymatic treatment, or in vivo by means ofrecombinant DNA technology. Such constructs may differ from thosedisclosed, for example, by virtue of one or more nucleotidesubstitutions, deletions or insertions, but substantially retain abiological activity of the construct or nucleic acid molecule of thisinvention.

The present invention further provides kits. Accordingly, in a fifthaspect the invention provides a kit used for improving the growthperformance of an animal comprising:

a). one or more anti-inflammatory agents;

b). a delivery device for said anti-inflammatory agents; and

c). instructions for use in the method of the invention.

Suitable buffering agents and ionic salts may also be included in thekit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the rate of gain over the first 4 weeks of the weaner phasefor pigs treated with IL-1ra or saline, in the presence or absence ofin-feed antibiotics. (Bars show group means and SEM).

FIG. 2 shows the rate of gain over weeks 5 and 6 of the weaner phase forpigs treated with IL-1ra or saline, in the presence or absence ofin-feed antibiotics. (Bars show group means and SEM).

FIG. 3 shows the rate of gain over the weaner (D7-D42) and grower phases(D79 and D93) in pigs treated with IL-1ra or saline, in the presence orabsence of in-feed antibiotics.

FIG. 4 shows the rate of gain over the finisher phase in pigs treatedwith IL-1ra or saline, in the presence or absence of in-feedantibiotics.

FIG. 5 shows the average weight at slaughter of pigs treated with salineor IL-1ra in the presence or absence of in-feed medication during theweaner phase.

FIG. 6 shows the average dressed weight (warm carcass weight) for pigstreated with saline or IL-1ra in the presence or absence of in-feedmedication during the weaner phase.

FIG. 7 shows the feed conversion ratio for pigs treated with saline orIL-1ra in the presence or absence of in-feed medication during theweaner phase. Feed conversion was calculated over the finisher phase(day 93-day 133).

FIG. 8 shows mean weights at the end of the weaner phase in pigs treatedwith either IL-1ra or saline and provided with zero, reduced and normallevels of antibiotic medication.

FIG. 9 shows the production losses during the weaner phase in terms ofincidence of weight loss and mortality in pigs treated with IL-1ra orsaline and provided with zero, reduced or normal levels of antibioticmedication.

FIG. 10 shows the total group weight at the end of the weaner phase inpigs treated with IL-1ra or saline and provided with zero, reduced ornormal levels of antibiotic medication.

FIG. 11 shows mean weights at the end of the grower phase in pigstreated with either IL-1ra or saline and provided with zero, reduced andnormal levels of antibiotic medication.

FIG. 12 shows mean weights at the end of the finisher phase in pigstreated with either IL-1ra or saline and provided with zero, reduced andnormal levels of antibiotic medication.

FIG. 13 shows mean P2 backfat measurements at slaughter in pigs treatedwith either IL-1ra or saline and provided with zero, reduced and normallevels of antibiotic medication.

FIG. 14 shows E. coli cultured from faeces collected from pigs treatedwith saline, IL-1ra or Apralan, for 5 days after initial challenge withE. coli. Data points show group means with standard errors.

FIG. 15 shows percentage reduction in total faecal culture scores over 5days after E. coli challenge, compared to saline controls, in pigstreated with either IL-1ra or Apralan.

FIG. 16 shows recordings of diarrhoea and wet faeces for 5 days after E.coli challenge in pigs treated with IL-1ra, saline or Apralan. Bars showthe total records for each group; the maximum records for any group is40.

FIG. 17 shows percentage reduction in clinical signs (faecal condition)of E. coli infection in pigs treated with IL-1ra or Apralan, compared tosaline controls.

FIG. 18 shows E. coli culture scores from samples taken in differentareas along the gastro-intestinal tract at post-mortem in pigs treatedwith IL-1ra, saline or Apralan. SI refers to the small intestine. Barsshow group means and standard errors.

FIG. 19 shows percentage reduction in E. coli culture scores at postmortem in pigs treated with either IL-1ra or Apralan, compared to salinetreated controls.

FIG. 20 shows total E. coli culture scores from all areas of thegastro-intestinal tract at post-mortem in pigs treated with IL-1ra,saline or Apralan. Bars show group means and standard errors.

FIG. 21 shows percentage reduction in the total levels of E. colipresent in the gut at post-mortem in pigs treated with IL-1ra orApralan, compared to saline controls.

FIG. 22 shows E. coli culture scores at post-mortem from the foregut andhindgut in pigs treated with IL-1ra, saline or Apralan. Bars indicategroups mean and standard error.

FIG. 23 shows percentage reduction in E. coli culture scores obtainedfrom the foregut and hindgut areas, in pigs treated with IL-1ra orApralan, compared to saline controls.

FIG. 24 shows Spirochaete culture scores from samples taken in differentareas along the gastro-intestinal tract at post-mortem in pigs treatedwith IL-1ra or saline. Bars indicate group mean.

FIG. 25 shows percentage reduction in spirochaete culture scores atpost-mortem for pigs treated with IL-1ra compared to saline controls.

FIG. 26 shows faecal condition at post-mortem in pigs treated withsaline or IL-1ra and challenged with swine dysentery.

FIG. 27 shows expression of mRNA for the pro-inflammatory cytokine TNFαin peripheral blood of pigs treated with IL-1ra or saline and challengedwith swine dysentery.

FIG. 28 shows expression of mRNA for the pro-inflammatory cytokine IL-8in peripheral blood of pigs treated with IL-1ra or saline and challengedwith swine dysentery.

FIG. 29 shows expression of mRNA for the pro-inflammatory cytokine IL-1in peripheral blood of pigs treated with IL-1ra or saline and challengedwith swine dysentery.

FIG. 30 shows average weight gain for 2 weeks in pigs treated withrecombinant IL-1ra, plasmid IL-1ra, the NSAID flunix, plasmid control orsaline control and subsequently challenged with App. Bars indicate groupmean and standard error.

FIG. 31 shows total weight gained during 14d challenge with App, in pigstreated with saline, flunix, recombinant IL-1ra, plasmid control orplasmid IL-1ra. Bars indicate group mean and standard error.

FIG. 32 shows daily rate of gain during 14d challenge with App, in pigstreated with saline, flunix, recombinant IL-1ra, plasmid control orplasmid IL-1ra. Bars indicate group mean and standard error.

FIG. 33 shows percentage change in weight gained compared to salinecontrols in pigs treated with either flunix or IL-1ra and subsequentlychallenged with App for 14d.

FIG. 34 shows percentage change in weight gained compared to salinecontrols and plasmid controls in pigs treated with IL-1ra plasmid andsubsequently challenged with App for l4d.

FIG. 35 shows levels of TNFα protein in the serum of pigs treated withsaline, flunix, recombinant IL-1ra, plasmid control of plasmid IL-1raand subsequently challenged with App. Bars indicate group mean andstandard error.

FIG. 36 shows expression of mRNA for the pro-inflammatory cytokine IL-6in peripheral blood in pigs, treated with saline, flunix, recombinantIL-1ra, plasmid control or plasmid IL-1ra and challenged with App. NSrefers to no sample for that time point. Bars indicate group mean andstandard error.

FIG. 37 shows presence of clinical signs of App disease over 30 visitsin the first week of challenge, in pigs treated with saline, flunix,recombinant IL-1ra, plasmid control or plasmid IL-1ra and challengedwith App. Bars indicate group miean and standard error. The maximumpossible score is 240.

FIG. 38 shows percentage reduction in clinical signs of disease in pigstreated with, flunix, recombinant IL-1ra, or plasmid IL-1ra andchallenged with App, compared to the relevant control groups.

FIG. 39 shows degree of pleurisy at necropsy, expressed as pleurisyscore (0-5) in pigs treated with saline, flunix, recombinant IL-1ra,plasmid control or plasmid IL-1ra and challenged with App. Bars indicategroup mean and standard error.

FIG. 40 shows percentage reduction in pleurisy in pigs treated withflunix, recombinant IL-1ra or plasmid IL-1ra and challenged with App,compared to the relevant controls.

FIG. 41 shows degree of pleuropneumonia at necropsy, expressed aspercentage of affected lung by weight, in pigs treated with saline,flunix, recombinant IL-1ra, plasmid control or plasmid IL-1ra andchallenged with App. Bars indicate group mean and standard error.

FIG. 42 shows percentage reduction in affected lung mass in pigs treatedwith flunix, recombinant IL-1ra or plasmid IL-1ra and challenged withApp, compared to the relevant controls.

FIG. 43 shows daily rate of gain in pigs treated with saline, low orhigh doses of IL-1ra, or IL-1ra+IL-4 (syn) during the first 10 days ofApp challenge. Bars indicate group mean and standard error.

FIG. 44 shows daily rate of gain in pigs treated with saline, low orhigh doses of IL-1ra, or IL-1ra+IL-4 (syn) during the second 10 days ofApp challenge. Bars indicate group mean and standard error.

FIG. 45 shows total weight gained in pigs treated with saline, low orhigh doses of IL-1ra, or IL-1ra+IL-4 (syn) during the total 21 days ofApp challenge. Bars indicate group mean and standard error.

FIG. 46 shows percentage improvement in weight gain compared to salinetreated controls over 21 days of App challenge in pigs treatedprophylactically with low or high doses of either IL-1ra, or IL-1ra+IL-4(syn).

FIG. 47 shows amount of lung affected by App lesions, described as apercentage of total lung weight in pigs treated with saline, low or highdoses of IL-1ra, or IL-1ra+IL-4 (syn) during the total 21 days of Appchallenge. Bars indicate group mean and standard error.

FIG. 48 shows pleurisy scores in lungs from pigs treated with saline,low or high doses of IL-1ra, or IL-1ra+IL-4 (syn) during the total 21days of App challenge. Bars indicate group mean and standard error.

FIG. 49 shows expression of mRNA for the pro-inflammatory cytokine,IL-8, in lung tissue taken at postmortem from pigs treated with saline,low or high doses of IL-1ra, or IL-1ra+IL-4 (syn) during the total 21days of App challenge. Bars indicate group mean and standard error.

FIG. 50 shows expression of mRNA for the pro-inflammatory cytokine,TNFα, in lung tissue taken at postmortem from pigs treated with saline,low or high doses of IL-1ra, or IL-1ra+IL-4 (syn) during the total 21days of App challenge. Bars indicate group mean and standard error.

FIG. 51 shows weight gained in week 2 of App challenge in pigssubsequently treated with IL-1ra at high or low doses, saline orexcenel. Bars indicate group means and standard error.

FIG. 52 shows feed intake in pigs challenged with App and subsequentlytreated with IL-1ra at high or low doses, saline or excenel. Barsindicate group means and standard error.

FIG. 53 shows feed conversion ratio pigs challenged with App andsubsequently treated with IL-1ra at high or low doses, saline orexcenel. Bars indicate group means and standard error.

FIG. 54 shows proliferative capacity of lymphocytes in response tostimulation with killed App, for pigs challenged with App andsubsequently treated with IL-1ra at high or low doses, saline orexcenel. Bars indicate group means and standard error.

FIG. 55 shows levels of mRNA for the pro-inflammatory cytokine IL-8,found in the lungs at post-mortem, in pigs challenged with App andsubsequently treated with IL-1ra at high or low doses, saline orexcenel. Bars indicate group means and standard error.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention employs, unless otherwiseindicated, conventional molecular biology, cellular biology, andrecombinant DNA techniques within the skill of the art. -Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Sambrook and Russell “Molecular Cloning: ALaboratory Manual” (2001) (Green Publishing, New York); Cloning: APractical Approach,” Volumes I and II (D. N. Glover, ed., 1985) (GreenPublishing, New York); “Oligonucleotide Synthesis” (M. J. Gait, ed.,1984); “Nucleic Acid Hybridisation” (B. D. Hames & S. J. Higgins, eds.,1985); “Antibodies: A Laboratory Manual” (Harlow & Lane, eds., 1988);“Transcription and Translation” (B. D. Hames & S. J. Higgins, eds.,1984); “Animal Cell Culture” (R. I. Preshney, ed., 1986); “ImmobilisedCells and Enzymes” (IRL Press, 1986); B. Perbal, “A Practical Guide toMolecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: aLaboratory Manual” (1989). Ausubel, F. et al., 1989-1999, “CurrentProtocols in Molecular Biology” (Green Publishing, New York).

Before the present methods and compositions are described, it isunderstood that this invention is not limited to the particularmaterials and methods described, as these may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims. It must be noted that as used herein and in theappended claims, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, a reference to “a soluble cytokine receptor” includes aplurality of such cytokine receptor, and a reference to “an antibiotic”is a reference to one or more antibiotics and equivalents thereof knownto those skilled in the art, and so forth. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any materials and methods similar orequivalent to those described herein can be used to practice or test thepresent invention, the preferred materials and methods are nowdescribed.

All publications mentioned herein are cited for the purpose ofdescribing and disclosing the protocols, reagents and vectors which arereported in the publications and which might be used in connection withthe invention. Nothing herein is to be construed as an admission thatthe invention is not entitled to antedate such disclosure by virtue ofprior invention.

It is to be understood that the methods and compositions of the presentinvention are useful for improving the “growth performance” of ananimal. The term “growth performance” is known in the art as a referenceto the criteria of growth rate and efficiency of feed use of an animal,and also a reference to the final weight of an animal, and the dressedweight and fat content of a carcass from the animal. The “growth rate”of an animal is the rate of unit gain in live weight of the animal and“efficiency of feed use” is the amount of feed required per unit gain inlive weight of the animal. The “final weight” of an animal is the weightof the animal at slaughter at a specified age and the “dressed weight”is the weight of a carcass from which viscera, feet, trotters or hooveshave been removed. The “fat content” is the amount of fat on a dressedcarcass. Methods for measuring the criteria of growth rate, efficiencyof feed use, final weight, and dressed weight and fat content of acarcass, are known to the skilled worker. See, for example, ManipulatingPig Production VI, VII & VIII. 1997, 1999 & 2001, Ed. P. D. Cranwell,Australian Pig Science Association, Werribee, Victoria, Australia.Growth rate is obtained from successive measurements of live weight overtime. Efficiency of feed use is obtained from successive measurements offeed disappearance and live weight over time. Carcass fat content istraditionally assumed from a measurement of back-fat thickness inmillimetres at the P2 position. Accordingly, in the present inventionthe term “growth performance” means an improvement in one or more of thecriteria of growth rate, efficiency of feed use, final or dressed weightand fat content of a carcass from an animal.

The term “animal” as used herein means any animal for which an increasein growth performance is desirable. For example, animals included in themammalian order Artiodactyls or in the avian class Aves.

Artiodactyls comprise approximately 150 living species distributedthrough nine families: pigs (Suidae), peccaries (Tayassuidae),hippopotamuses (Hippopotamidae), camels (Camelidae), chevrotains(Tragulidae), giraffes and okapi (Giraffidae), deer (Cervidae),pronghorn (Antilocapridae), and cattle, sheep, goats and antelope(Bovidae). Many of these animals are used as feed animals in variouscountries. More importantly, with respect to the present invention, manyof the economically important animals such as goats, sheep, cattle andpigs have very similar biology and share a high degree of genomichomology. More importantly, it is well known that certain animals suchas goats and sheep and horses and donkeys can interbreed.

The terms “bird” and “avian” as used herein, are intended to include allavian species, including, but not limited to, chickens, turkeys, ducks,geese, quail, and pheasant which are commercially raised for eggs ormeat. This term also includes both males and females of any avianspecies. Accordingly, the terms “bird” and “avian” are particularlyintended to encompass hens, cocks and drakes of chickens, turkeys,ducks, geese, quail and pheasant. Chickens and turkeys are preferred.

All Artiodactyls have similar inflammatory systems which includescytokine systems, in that they posses, for example, interleukins,GM-CSF, interferon's α, β and γ and their respective receptors. In mostspecies the genes coding for these cytokines map to particular regionson certain chromosomes. For example, in humans, the interleukin 5 genemaps to chromosome 5q23-31 in the same area as genes encoding GM-CSF,M-CSF, IL-3 and IL-4. More importantly, many of the cytokines and theirreceptors have high degrees of amino acid sequence homologies betweendifferent species. For example, it is well known in the art that porcineinterleukin 5 shares as much as 90% of its amino acids with animals suchas bovine, ovine and equine (See, for example, Sylvin et al. (2000),Immunogenetics, 51: 59-64). Indeed, even species as distinct as mice andhumans share as much as 70% amino acid sequence identities (See, forexample, Dictionary of Cytokines (1995), Horst. Ibelgaufts, VCHPublishers, Weinheim). Furthermore, it is known that human IL-10 has asignificant degree of sequence homology with bovine, murine, and ovineIL-10 (Dutia et al. (1994) Gene; 149:393-4).

It is also well known in the art that a number of cytokines have speciescross-reactivity. For example, IL-4 has some cross-species reactivity,while IL-5 has a high level of cross-species reactivity Dictionary ofCytokines (1995), Horst Ibelgaufts, VCH Publishers, Weinheim. However,it should be noted that the cross-reactivity described in the prior artliterature relates to in-vitro assays and some in-vivo experiments, butdoes not relate to growth performance.

Cytokines are also known to regulate the expression of cytokinereceptors, either in a stipulatory or inhibitory manner, therebycontrolling the biological activities of cytokines by other cytokines.Some cytokines share common receptor subunits which may have aregulatory effect. For example, the GM-CSF receptor shows significanthomologies with other receptors for Hematopoietic growth factors,including IL-2-β, IL-3, IL-6, IL-7, Epo and the Prolactin receptors(See, for example, cytokines Online PathfinderEncyclopaedia—www.copewithcytokines.de). It is also known that IL-3 iscapable of upregulating the expression of GM-CSF receptors on mousemacrophages, IL-3 also upregulates IL-1 receptor expression on human andmurine bone marrow cells, IL-4 upregulates IL-1 type 1 receptorexpression and down regulate IL-2 receptor expression. Furthermore, IL-7upregulates IL-4 Receptor expression, and TNFα upregulates both IL-3 andGM-CSF Receptor expression (Dictionary of Cytokines (1995), HorstIbelgaufts, VCH Publishers, Weinheim).

In a similar fashion to Artiodactyls, birds also have common cytokinesystems, including interleukins. Accordingly, the term “avian cytokinereceptor,” or “bird cytokine receptor” as used herein, means anycytokine receptor corresponding to an cytokine produced by any avianspecies.

Given the level of common ancestry and biology for many of the feedanimals, the high degree of amino acid sequence homology for cytokinesand other inflammatory processes across a number of species such ascattle, sheep, goats and pigs, and the level of cross-species reactivityof the cytokines a person skilled in the art would appreciate that thecompositions and methods disclosed herein are applicable for all feedanimals and for all cytokine receptors.

It will be further appreciated by those skilled in the art that thecompositions and methods disclosed herein may be directly extrapolatedto encompass other aspects of the invention. For example, data arepresented for specific cytokine receptor antagonists; however, these arenot to be construed to be limiting on the invention. Indeed, thecytokine receptor antagonists disclosed were specifically chosen toillustrate the breadth of the invention. For example, many cytokinesshare receptors or receptor subunits. For example, IL-3, IL-5 and GM-CSFshare a receptor subunit (Dictionary of -Cytokines (1995), HorstIbelgaufts, VCH Publishers, Weinheim). IL-4 shares a common subunit withIL-2 and IL-7 (Dictionary of Cytokines (1995), Horst Ibelgaufts, VCHPublishers, Weinheim). Some cytokines have similar gene structures andare clustered on the one chromosome eg IL-3, IL-4, IL-5, GM-CSF andIL-13 in humans and mice (Dictionary of Cytokines (1995), HorstIbelgaufts, VCH Publishers, Weinheim).

All of the foregoing is illustrative of the breadth of the presentlydisclosed invention with respect the types of animals encompassed.However, it will also be readily seen that the term “cytokine receptor”or “cytokine receptor antagonists” is also to be construed broadly andnot limited to the experimental data disclosed. For example, the term“cytokine receptor” includes one or more of IL-1 receptor, IL-6receptor, TGF-β receptor, IL-11 receptor, IL-18 receptor, IL-12receptor, IL-17 receptor, LIF receptor, IPN-γ receptor IL-8 receptor,TNF-α receptor and GM-CSF receptor, in soluble form. The term “receptorantagonist” includes IL-1ra, IL-4ra, IL-8ra, GM-CSFra, IL-6ra orTNF-αra.

In one particularly preferred embodiment the initial step in the methodof the invention involves the administration of a growth promotingamount one or more anti-inflammatory agents to an animal.

The term “anti-inflammatory agent” as used herein refers to any compoundor composition which is capable of reducing inflammation. For example,soluble cytokine receptors, cytokine receptor antagonists, cytokineinhibitory factors or biologically active fragments thereof which havean anti-inflammatory effect may be used. Alternatively, ananti-inflammatory agent such as diclofenac, diflunisal, etodolac,flunix, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin,ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone,naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam,tiaprofenic or tolmetin may be used. Corticosteroid drugs are also knownas powerful anti-inflammatory agents that are used widely to suppressthe harmful effects of immune responses. Accordingly, in one embodimentcorticosteroid drugs may used.

In a further embodiment, the term “anti-inflammatory agent” includes anycompound or composition which increase the number of soluble receptorsfor pro-inflammatory cytokines.

As discussed above, growth performance is measurable; however, why thereis an increase in growth performance is a little more complex. While notwishing to be bound by any particular theory or hypothesis, theapplicant believes that the administration of an anti-inflammatory agentacts in a number of complementary ways that result in the improvedgrowth performance. For example, the applicant has found that byimproving the immunity of feed animals, stock losses are avoided andconsequently growth performance improves. Thus, the present inventionprovides a method of reducing the susceptibility of an animal toinfection. The method is useful for reducing susceptibility to infectionby bacteria, virus or parasite.

The applicant has also found that the administration of one or moreanti-inflammatory agents together with one or more antibiotics alsoimproves the growth performance of an animal while reducing the totalamount of antibiotic used. It is believed that antibiotic limits themicrobial load in the animal to a threshold level at which theadministered anti-inflammatory agents is then capable of exerting aneffect on growth performance.

Accordingly, the applicant believes that rather than functioning as agrowth promoter per se, although this may be possible, it will beunderstood that administration of the anti-inflammatory agents may causeimproved growth performance by activating the humoral and cellular armsof the immune response which are capable of being activated by theanti-inflammatory agents.

As used herein, the term “Growth promoting amounts is meant an amount ofan anti-inflammatory agent of the present invention effective to yieldan increase in growth performance as defined above. For example,increased growth rate, efficiency of feed use, increased final weight,increased carcass dressed weight or reduced fat content.

As used herein, the term “administration” refers to the mode of deliveryof a composition of the invention. The term also refers to the dosage ofa composition. Depending upon the activity of an anti-inflammatory agentand age and body weight of an animal, the manner of administration anddosage of an anti-inflammatory agent will vary. It will be understoodthat the specific dose level for any particular animal will depend upona variety of factors including the activity of the specificanti-inflammatory agent employed, the age, body weight, general health,sex, diet, time of administration, route of administration, rate ofexcretion and anti-inflammatory agent or antibiotic combination.However, generally the preferred route of administration is selectedfrom the group consisting of oral, topical and parenteraladministration.

Parenteral administration includes subcutaneous injections, aerosol,intravenous, intramuscular, intrathecal, injection or infusiontechniques and encapsulated cells.

As used herein, the term “upregulate” or “upregulating” refers toinducing an increase in production, secretion or availability (and thusan increase in the concentration) of a protein or peptide. A method ofupregulating endogenous anti-inflammatory agent in an animal or avianthus refers to a method of inducing an increase in the production,secretion or availability of anti-inflammatory agent in the animal oravian, as compared to an untreated animal or avian.

The term “endogenous” means originating within the subject, cell, orsystem being studied. Accordingly, supplementing the endogenous levelsof an anti-inflammatory agent means that a compound or compounds is/areadministered to an animal such that the total amount of ananti-inflammatory agent in the animal is higher than normally present.Increasing the endogenous levels of an anti-inflammatory agent meansthat a compound or compounds is/are administered to an animal where thecompound or compounds increase the production of an anti-inflammatoryagent by an animals cells or tissue, thereby effectively increasing thetotal amount of an anti-inflammatory agent in the animal. The endogenouslevels of an anti-inflammatory agent may also be effectively increasedby decreasing the turn over rate of a the anti-inflammatory agent. Forexample, a compound or compounds of the invention when administered toan animal may decrease the rate of proteolysis of endogenousanti-inflammatory agents by inhibiting the effect of proteolyticenzymes.

Many substances are able to stimulate upregulation of endogenousanti-inflammatory agents such as cytokine receptors, IL-4 and IL-16 orcytokine receptor antagonists. For example, as shown in InternationalPatent Application No WO93/18783, IL-10 upregulates the expression onIL-1 receptor antagonist. Furthermore, compounds such 1,8-napthosultamsubstituted compounds or quinoxaline compounds are known to upregulatecytokine receptor antagonists such as IL-8. See, for example,International Patent Application Nos WO99/36070 and WO99/42461 hereinincorporated by reference.

An alternative process of reducing or ameliorating the effects ofpro-inflammatory cytokine such as IL-1, is by removing these fromcirculation. For example, it is well known that there are factors thatare capable of binding to ligands thereby preventing them from bindingtheir receptors. TNF blocking factor and TNF-α inhibitor, for example,discussed supra are known to bind to TNF.

The term “biologically active fragment” refers to a segment of ananti-inflammatory agent having a biological or physiological effect inan animal that is substantially similar to the entire or completeanti-inflammatory agent from which it is derived. For example, abiologically active fragment of IL-1 receptor antagonist may be anyportion of IL-1 receptor antagonist having greater than about 5 aminoacid residues which either comprises a biologically active site orwherein the portion retains IL-1 receptor antagonist activity. Forexample, if the IL-1 receptor antagonist portion retains the ability tobind to the IL-1 receptor as discussed above then this portion is a“biologically active fragment” of IL-1 receptor antagonist. Typically,such a fragment of IL-1 receptor antagonist is one capable ofcompetitively inhibiting the binding of IL-1 to the IL-1 receptor.

It follows that a fragment of IL-1ra sufficient for providing some orall of IL-1ra function, or any other molecule sufficient for providingsome or all of IL-1ra function, may be administered in the method,rather than IL-1ra. For example, such a fragment or molecule is capableof providing some or all of the function of IL-1ra including blockingIL-6 and IL-8 production. Typically, such a fragment or molecule is onecapable of competitively inhibiting the binding of IL-1ra and/or IL-1 tothe IL-1 receptor. Thus, in one embodiment, the invention comprisesadministering a fragment of IL-1ra sufficient for providing some or allof IL-1ra function, or a molecule sufficient for providing some or allof IL-1ra function.

Amino acid sequence variants of the amino acid sequence of a solublecytokine receptor, cytokine receptor antagonist, cytokine inhibitoryfactor or biologically active fragment thereof are also encompassed. Forexample, where one or more amino acid residues are added at the N— orC-terminus of, or within, the soluble. cytokine receptor, cytokinereceptor antagonist, cytokine inhibitory factor or biologically activefragment thereof sequence or its fragments as defined above. Amino acidsequence variants of a soluble cytokine receptor, cytokine receptorantagonist, cytokine inhibitory factor sequence or their fragments asdefined above, wherein one or more amino acid residues of the sequenceor fragment thereof are deleted, and optionally substituted by one ormore amino acid residues; and derivatives of the above soluble cytokinereceptor, cytokine receptor antagonist, cytokine inhibitory factor orbiologically active fragment thereof, wherein an amino acid residue hasbeen covalently modified so that the resulting product is anon-naturally occurring amino acid. Again all of these variants ofsoluble cytokine receptor, cytokine receptor antagonist, cytokineinhibitory factor or biologically active fragment thereof areencompassed by the term “biologically active fragment” as long as thevariants retain the biological activity of the entire soluble cytokinereceptor, cytokine receptor antagonist, cytokine inhibitory factor orbiologically active fragment thereof from which it is derived.

As used herein, a “pharmaceutical carrier, adjuvant or vehicle” is apharmaceutically acceptable solvent, suspending agent or vehicle fordelivering the anti-inflammatory agent and/or antibiotic to the animal.The carrier may be liquid or solid and is selected with the plannedmanner of administration in mind.

The term “substantially homologous” can refer both to nucleic acidand/or amino acid sequences, means that a particular subject sequence,for example, a mutant sequence, varies from a reference sequence by oneor more substitutions, deletions, or additions, the net effect of whichdoes not result in an adverse functional dissimilarity-between referenceand subject sequences. For purposes of the present invention, sequenceshaving equivalent biological activity and equivalent expressioncharacteristics are considered substantially homologous. Sequenceshaving lesser degrees of identity, comparable bioactivity, andequivalent expression characteristics are considered equivalents.

“Microbial” refers to recombinant proteins made in bacterial, fungal(e.g., yeast), viral (e.g. baculovirus), or plant expression systems. Asa product, “recombinant microbial” defines an animal protein essentiallyfree of native endogenous substances and unaccompanied by associatednative glycosylation. Protein expressed in most bacterial cultures,e.g., E. coli, will be free of glycosylation modifications; proteinexpressed in yeast and insect cells will have a glycosylation patterndifferent from that expressed in mammalian cells.

A “nucleic acid molecule” or “polynucleic acid molecule” refers hereinto deoxyribonucleic acid and ribonucleic acid in all their forms, ie.single and double-stranded DNA, cDNA, mRNA, and the like.

A “double-stranded DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thymine, or cytosine) in itsnormal, double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (eg. restriction fragments),viruses, plasmids, and chromosomes. In discussing the structure ofparticular double-stranded DNA molecules, sequences may be describedherein according to the normal convention of giving only the sequence inthe 5′ to 3′ direction along the non-transcribed strand of DNA (ie. thestrand having a sequence homologous to the mRNA).

A DNA sequence “corresponds” to an amino acid sequence if translation ofthe DNA sequence in accordance with the genetic code yields the aminoacid sequence (ie. the DNA sequence “encodes” the amino acid sequence).

One DNA sequence “corresponds” to another DNA sequence if the twosequences encode the same amino acid sequence.

Two DNA sequences are “substantially similar” when at least about 85%,preferably at least about 90%, and most preferably at least about 95%,of the nucleotides match over the defined length of the DNA sequences.Sequences that are substantially similar can be identified in a Southernhybridization experiment, for example under stringent conditions asdefined for that particular system. Defining appropriatehybridization-conditions is within the skill of the art. See eg.Sambrook et al., DNA Cloning, vols. I, II and III. Nucleic AcidHybridization. However, ordinarily, “stringent conditions” forhybridization or annealing of nucleic acid molecules are those that

(1) employ low ionic strength and high temperature for washing, forexample, 0.015M NaCl/0.0015M sodium citrate/0.1% sodium dodecyl sulfate(SDS) at 50° C., or (2) employ during hybridization a denaturing agentsuch as formamide, for example, 50% (vol/vol) formamide with 0.1% bovineserum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.

Another example is use of 50% formamide, 5×SSC (0.75M NaCl, 0.075Msodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50μg/mL), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC and 0.1% SDS.

A “heterologous” region or domain of a DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the gene will usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous regionis a construct where the coding sequence itself is not found in nature(eg. a cDNA where the genomic coding sequence contains introns orsynthetic sequences having codons different than the native gene).Allelic variations or naturally-occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

A “coding sequence” is an in-frame sequence of codons that correspond toor encode a protein or peptide sequence. Two coding sequences correspondto each other if the sequences or their complementary sequences encodethe same amino acid sequences. A coding sequence in association withappropriate regulatory sequences may be transcribed and translated intoa polypeptide in vivo. A polyadenylation signal and transcriptiontermination sequence will usually be located 3′ to the coding sequence.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. A coding sequence is “under the control” ofthe promoter sequence in a cell when RNA polymerase which binds thepromoter sequence transcribes the coding sequence into mRNA, which isthen in turn translated into the protein encoded by the coding sequence.

For the purposes of the present invention, the promoter sequence isbounded at its 3′ terminus by the translation start codon of a codingsequence, and extends upstream to include the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter sequence will be found a transcriptioninitiation site (conveniently defined by mapping with nuclease S1), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. Bukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes; prokaryotic promoterscontain Shine-Delgarno sequences in addition to the −10 and −35consensus sequences.

A cell has been “transformed” by exogenous DNA when such exogenous DNAhas been introduced inside the cell wall. Exogenous DNA may or may notbe integrated (covalently linked) to chromosomal DNA making up thegenome of the cell. In prokaryotes and yeast, for example, the exogenousDNA may be maintained on an episomal element such as a plasmid. Withrespect to eukaryotic cells, a stably transformed cell is one in whichthe exogenous DNA is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

“Integration” of the DNA may be effected using non-homologousrecombination following mass transfer of DNA into the cells usingmicroinjection, biolistics, electroporation or lipofection. Alternativemethods such as homologous recombination, and or restriction enzymemediated integration (REMI) or transposons are also encompassed, and maybe considered to be improved integration methods.

A “clone” is a population of cells derived from a single cell or commonancestor by mitosis.

“Cell,” “host cell, ” “cell line,” and “cell culture” are usedinterchangeably herewith and all such terms should be understood toinclude progeny. A “cell line” is a clone of a primary cell that iscapable of stable growth in vitro for many generations. Thus the words“transformants” and “transformed cells” include the primary subject celland cultures derived therefrom, without regard for the number of timesthe cultures have been passaged. It should also be understood that allprogeny might not be precisely identical in DNA content, due todeliberate or inadvertent mutations.

Vectors are used to introduce a foreign substance, such as DNA, RNA orprotein, into an organism. Typical vectors include recombinant viruses(for DNA) and liposomes (for protein). A “DNA cloning vector” is anautonomously replicating DNA molecule, such as plasmid, phage or cosmid.Typically the DNA cloning vector comprises one or a small number ofrestriction endonuclease recognition sites, at which such DNA sequencesmay be cut in a determinable fashion without loss of an essentialbiological function of the vector, and into which a DNA fragment may bespliced in order to bring about its replication and cloning. The cloningvector may also comprise a marker suitable for use in the identificationof cells transformed with the cloning vector.

An “expression vector” is similar to a DNA cloning vector, but containsregulatory sequences which are able to direct protein synthesis by anappropriate host cell. This usually means a promoter to bind RNApolymerase and initiate transcription of mRNA, as well as ribosomebinding sites and initiation signals to direct translation of the mRNAinto a polypeptide. Incorporation of a DNA sequence into an expressionvector at the proper site and in correct reading frame, followed bytransformation of an appropriate host cell by the vector, enables theproduction of mRNA corresponding to the DNA sequence, and usually of aprotein encoded by the DNA sequence.

For the purposes of the present invention, the promoter sequence isbounded at its 3′ terminus by the translation start codon of a codingsequence, and extends upstream to include the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter sequence will be found a transcriptioninitiation site (conveniently defined by mapping with nuclease Si), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase.

An “exogenous” element is one that is foreign to the host cell, or ishomologous to the host cell but in a position within the host cell inwhich the element is ordinarily not found.

“Digestion” of DNA refers to the-catalytic cleavage of DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes or restriction endonucleases, and the siteswithin DNA where such enzymes cleave are called restriction sites. Ifthere are multiple restriction sites within the DNA, digestion willproduce two or more linearized DNA fragments (restriction fragments).The various restriction enzymes used herein are commercially available,and their reaction conditions, cofactors, and other requirements asestablished by the enzyme manufacturers are used. Restriction enzymesare commonly designated by abbreviations composed of a capital letterfollowed by other letters representing the microorganism from which eachrestriction enzyme originally was obtained and then a number designatingthe particular enzyme. In general, about 1 μg of DNA is digested withabout 1-2 units of enzyme in about 20 μl of buffer solution. Appropriatebuffers and substrate amounts for particular restriction enzymes arespecified by the manufacturer, and/or are well known in the art.

“Recovery” or “isolation” of a given fragment of DNA from a restrictiondigest typically is accomplished by separating the digestion products,which are referred to as “restriction fragments,” on a polyacrylamide oragarose gel by electrophoresis, identifying the fragment of interest onthe basis of its mobility relative to that of marker DNA fragments ofknown molecular weight, excising the portion of the gel that containsthe desired fragment, and separating the DNA from the gel, for exampleby electroelution.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double-stranded DNA fragments. Unless otherwise specified, ligationis accomplished using known buffers and conditions with 10 units of T4DNA ligase per 0.5 μg of approximately equimolar amounts of the DNAfragments to be ligated.

“Oligonucleotides” are short-length, single- or double-strandedpolydeoxynucleotides that are chemically synthesized by known methods(involving, for example, triester, phosphoramidite, or phosphonatechemistry), such as described by Engels, et al., Agnew. Chem. Int. Ed.Engl. 28:716-734 (1989). They are then purified, for example, bypolyacrylamide gel electrophoresis.

“Polymerase chain reaction,” or “PCR,” as used herein generally refersto a method for amplification of a desired nucleotide sequence in vitro,as described in U.S. Pat. No. 4,683,195. In general, the PCR methodinvolves repeated cycles of primer extension synthesis, using twooligonucleotide primers capable of hybridizing preferentially to atemplate nucleic acid. Typically, the primers used in the PCR methodwill be complementary to nucleotide sequences within the template atboth ends of or flanking the nucleotide sequence to be amplified,although primers complementary to the nucleotide sequence to beamplified also may be used. Wang, et al., in PCR Protocols, pp. 70-75(Academic Press, 1990); Ochman, et al., in PCR Protocols, pp. 219-227;Triglia, et al., Nucl. Acids Res. 16:8186 (1988).

“PCR cloning” refers to the use of the PCR method to amplify a specificdesired nucleotide sequence that is present amongst the nucleic acidsfrom a suitable cell or tissue source, including total genomic DNA andcDNA transcribed from total cellular RNA. Prohman, et al., Proc. Nat.Acad. Sci. USA 85:8998-9002 (1988); Saiki, et al., Science 239:487-492(1988); Mullis, et al., Meth. Enzymol. 155:335-350 (1987).

A “vector” or “construct” refers to a plasmid or virus or genomicintegration comprising a transcriptional unit with (1) a genetic elementor elements having a regulatory role in gene expression, for example,promoters or enhancers, (2) a structural or coding sequence which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription initiation and termination sequences. Structural unitsintended for use in yeast or eukaryotic expression systems would includea leader sequence enabling extracellular secretion of translated proteinby a host cell. Alternatively, where recombinant protein is expressedwithout a leader or transport sequence, it may include an N-terminalmethionine residue. This residue may or may not be subsequently cleavedfrom the expressed recombinant protein to provide a final product.Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, and a promoter derived from a highly-expressed gene to inducetranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably, a leader sequencecapable of directing secretion of translated protein into theperiplasmic space or extracellular medium. Optionally, the heterologoussequence can encode a fusion protein including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. Preferred recombinant expression vectors of the invention areviral vectors (eg. porcine adenoviral vector, mammalian cells (eg.porcine cells), plant cells and bacterial cells.

The term “immune response” is meant to refer to any response to anantigen or antigenic determinant by the immune system of a vertebratesubject. Exemplary immune responses include humoral immune responses(e.g. production of antigen-specific antibodies) and cell-mediatedimmune responses (e.g. lymphocyte proliferation), as defined hereinbelow.

The term “systemic immune response” is meant to refer to an immuneresponse in the lymph node-, spleen-, or gut-associated lymphoid tissueswherein cells, such as B lymphocytes, of the immune system aredeveloped. For example, a systemic immune response can comprise theproduction of serum IgG's. Further, systemic immune response refers toantigen-specific antibodies circulating in the blood stream andantigen-specific cells in lymphoid tissue in systemic compartments suchas the spleen and lymph nodes. In contrast, the gut-associated lymphoidtissue (GALT) is a component of the mucosal immune system sinceantigen-specific cells that respond to gut antigens/pathogens areinduced and detectable in the GALT.

As cytokine receptor and cytokine receptor antagonist are endogenouslyexpressed in all feed animal species and that many of these have a highdegree of cross-reactivity, it follows that cytokine receptors andcytokine receptor antagonists from one species may be administered toanimals of a different species and vice versa. For example, when theanimal is a pig, human cytokine receptors such as IL-1 receptor may beused in the disclosed methods. There is no requirement that theparticular cytokine receptor or cytokine receptor antagonist isidentical to the cytokine receptor or cytokine receptor antagonist whichis endogenously expressed in the animal.

The methods of this invention involve in one embodiment:

(1) The administration of one or more anti-inflammatory agents, priorto, together with, or subsequent to the administration of one or moreantibiotics; or

(2) The administration of a composition comprising 30 one or moreanti-inflammatory agents and one or more antibiotics.

(3) The administration of one or more anti-inflammatory agents withoutany antibiotics.

The anti-inflammatory agent(s) or composition(s) 35 of the invention maybe administered orally, topically, or parenterally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles. The term parenteral asused herein includes subcutaneous injections, aerosol, intravenous,intramuscular, intrathecal, intracranial, injection or infusiontechniques.

The present invention also provides suitable topical, oral, andparenteral pharmaceutical formulations for use in the novel methods ofimproving growth performance of the present invention. The compositionsof the present invention may be administered orally as tablets, aqueousor oily suspensions, lozenges, troches, powders, granules, emulsions,capsules, syrups or elixirs. The composition for oral use may containone or more agents selected from the group of sweetening agents,flavouring agents, colouring agents and preserving agents in order toproduce pharmaceutically elegant and palatable preparations. The tabletscontain the active ingredient in admixture with non-toxicpharmaceutically acceptable carriers, adjuvants or vehicles which aresuitable for the manufacture of tablets.

These carriers, adjuvants or vehicles may be, for example, (1) inertdiluents, such as calcium carbonate, lactose, calcium phosphate orsodium phosphate; (2) granulating and disintegrating agents, such ascorn starch or alginic acid; (3) binding agents, such as starch,gelatine or acacia; and (4) lubricating agents, such as magnesiumstearate, stearic acid or talc. These tablets may be uncoated or coatedby known techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. Coating may also beperformed using techniques described in the U.S. Pat. Nos. 4,256,108;4,160,452; and 4,265,874 to form osmotic therapeutic tablets for controlrelease.

The anti-inflammatory agents as well as the antibiotics useful in themethods of the invention can be administered, for in vivo application,parenterally by injection or by gradual perfusion over timeindependently or together. Administration may be intravenously,intra-arterial, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, anti-microbials, anti-oxidants, chelating agents, growthfactors and inert gases and the like.

The invention includes various compositions useful for improving growthperformance. The compositions according to one embodiment of theinvention are prepared by bringing one or more anti-inflammatory agents,with or without one or more antibiotics into a form suitable foradministration to an animal using carriers, adjuvants, vehicles oradditives.

Antibiotics suitable for use in this aspect of the invention are thoseconventionally used in animal husbandry as an additive to animal waterand/or feed and for limiting microbial load in the animal. Examples ofthese antibiotics include lincomycin, spectinomycin and amoxycillin. Adetailed analysis of antibiotic usage for food-producing animals inAustralia is described in “The use of antibiotics in food-producinganimals: antibiotic-resistant bacteria in animals and humans”. Report ofthe Joint Expert Advisory Committee on Antibiotic Resistance (JETACAR),Commonwealth of Australia, 1999.

An antibiotic can be administered to the animal in an amount that is thesame as the amount which would be conventionally administered to theanimal for the purpose of decreasing microbial load in the animal. Theseamounts of antibiotic are known to the skilled worker and referred to inJETACAR above.

Frequently used carriers, adjuvants or vehicles include magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars, talc,milk protein, gelatine, starch, vitamins, cellulose and its derivatives,animal and vegetable oils, polyethylene glycols and solvents, such assterile water, alcohols, glycerol and polyhydric alcohols. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial, anti-oxidants, chelating agents and inert gases. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in Remington's PharmaceuticalSciences, 15th ed. Easton: Mack Publishing Co., 1405-1412, 1461-1487(1975) and The National Formulary XIV., 14th ed. Washington: AmericanPharmaceutical Association (1975), the contents of which are herebyincorporated by reference. The pH and exact concentration of the variouscomponents of the pharmaceutical composition are adjusted according toroutine skills in the art. See Goodman and Gilman's The PharmacologicalBasis for Therapeutics (7th ed.).

The pharmaceutical compositions according to the invention may beadministered locally or systemically in a growth promoting amount.Amounts effective for this use will, of course, depend on theanti-inflammatory agent and the weight and general state of the animal.Typically, dosages used in vitro may provide useful guidance in theamounts useful for in situ administration of the compositions. Variousconsiderations are described, eg., in Langer, Science, 249: 1527,(1990). Formulations for oral use may be in the form of hard gelatinecapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin.They may also be in the form of soft gelatine capsules wherein theactive ingredient is mixed with water or an oil medium, such as peanutoil, liquid paraffin or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspension. Suchexcipients may be (1) suspending agent such as sodium carboxymethylcellulose, methyl cellulose, hydroxypropylmethylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2)dispersing or wetting agents which may be (a) naturally occurringphosphatide such as lecithin; (b) a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate; (c) acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethylenoxycetanol; (d) a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand hexitol such as polyoxyethylene sorbitol monooleate, or (e) acondensation product of ethylene oxide with a partial ester derived fromfatty acids and hexitol anhydrides, for example polyoxyethylene sorbitanmonooleate.

The compositions may be in the form of a sterile injectable aqueous oroleagenous suspension. This suspension may be formulated according toknown methods using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Anti-inflammatory agents and compositions of the invention may also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine, or phosphatidylcholines.

Dosage levels of the anti-inflammatory agents or compositions of thepresent invention are of the order of about 1 microgram to about 50microgram per kilogram body weight, with a preferred dosage rangebetween about 5 microgram to about 20 microgram per kilogram body weightper—dose (could be multiple or single) (from about 100 micrograms toabout 500 micrograms per animal per dose). The amount ofanti-inflammatory agent that may be combined with the carrier materialsto produce a single dosage will vary depending upon the animal and theparticular mode of administration. For example, a formulation intendedfor intravenous administration to a pig may contain about 20 μg to 1 gof anti-inflammatory agent with an appropriate and convenient amount ofcarrier material which may vary from about 5 to 95 percent of the total-composition. Dosage unit forms will generally contain between fromabout 5 μg to 500 mg of anti-inflammatory agent.

It will be understood, however, that the specific dose level for anyparticular animal will depend upon a variety of factors including theactivity of the specific anti-inflammatory agent employed, the age, bodyweight, general health, diet, time of administration, route ofadministration, rate of excretion and drug combination.

In one particularly preferred embodiment of the present invention theanti-inflammatory agent or agents are expressed in vivo rather thanadministered exogenously. For example, by inserting a structural DNAsequence encoding an anti-inflammatory agent together with suitabletranslation initiation and termination signals in operable reading phasewith a functional promoter an expression vector is created which wouldbe able to express the anti-inflammatory agent in vivo. The vector willcomprise one or more phenotypic selectable markers and an origin ofreplication to ensure amplification within the host. Suitableprokaryotic hosts for transformation include E. coli, Bacillus subtilis,Salmonella typhimurium and various species within the generaPseudomonas, Streptomonas; and Staphylococcus, although others may alsobe employed as a matter of choice. Following transformation of asuitable host strain and expression, the cells are cultured for anadditional period. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. Various mammalian cell culturesystems can also be employed to express recombinant protein. Examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described by Gluzman, Cell 23:175 (1981), and other celllines capable of expressing a compatible vector, for example, the C127,3T3, CHO, HeLa and BHK cell lines and of course porcine cells. Mammalianexpression vectors will comprise an origin of replication, a suitablepromoter, and enhancer, and also any necessary ribosome binding sites,polyadenylation sites, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required non-transcribed genetic elements. Recombinantprotein produced in bacterial culture is usually isolated by initialextraction from cell pellets, followed by one or more salting out,aqueous ion exchange or size exclusion chromatography steps. Proteinrefolding steps can be used, as necessary, in completing configurationof the mature protein. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of proteins can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents. Use of an expression systemthat expresses a tag sequence for purification would simplifypurification. Recombinant expression systems as defined herein willexpress heterologous protein upon induction of the regulatory elementslinked to the DNA segment or synthetic gene to be expressed. Cell-freetranslation systems can also be employed to produce porcine cytokinesusing RNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Maniatis, Molecular Cloning: ALaboratory Manual, (Cold Spring Harbor, N.Y., 1985), the disclosure ofwhich is hereby incorporated by reference.

The nucleic acid encoding a particular anti-inflammatory agent isadvantageously in the form of plasmid DNA or a viral vector (whichvector is derived from an adenovirus, retrovirus, poxvirus, inparticular from a vaccinia virus or an MVA virus, herpes virus,adenovirus-associated virus, etc.). The nucleic acid encoding aparticular anti-inflammatory agent is transported by means of aninfectious viral particle or in the form of a synthetic vector (cationiclipid, liposome, cationic polymer, etc.) or an engineered cell (cellwhich is transfected or transduced with the said nucleic acid) ornon-engineered cell (which naturally contains the said nucleic acid).

According to an additionally preferred variant, the nucleic acid ofinterest is carried by an adenoviral vector which is defective forreplication (unable to replicate autonomously in a host cell). Thetechnology of adenoviruses is described in the state of the art (see,for example, Graham and Prevec in Methods in Molecular Biology, 1991,vol 7, pp. 109-128, ed E. J. Murey, The Human Press Inc).Advantageously, the adenoviral vector which is used within the contextof the present invention is derived from the genome of an adenovirus,comprises at least the ITRs (inverted terminal repeats) and anencapsidation sequence and lacks all or part of the E1 adenoviralregion. In addition, it can lack all or part of the E3 adenoviralregion. However, according to an advantageous embodiment, preference isgiven to retaining the part of the E3 region which encodes polypeptides,in particular the glycoprotein gp19 k (Gooding et al., Critical Reviewof Immunology, 1990, 10: 53-71), which make it possible to escape theimmune system of the host. Furthermore, the vector can containadditional deletions or mutations which affect, in particular, all orpart of one or more regions selected from the E2, E4, L1, L2, L3, L4 andL5 regions (see, for example, international application WO 94/28152). Inorder to illustrate this point, mention may be made of thetemperature-sensitive mutation which affects the DBP (standing forDNA-binding protein) gene of the E2 A region (Ensinger et al., J.Virol., 1972, 10: 328-339). Another variant, or attractive combination,consists in deleting the E4 region with the exception of the sequenceswhich encode open reading frames (ORFs) 6 and 7 (these limited deletionsdo not require the E4 function to be complemented;. Ketner et al.,Nucleic Acids Res., 1989, 17: 3037-3048). Preferably, the gene(s) ofinterest is/are inserted into the vector in place of the deletedadenoviral regions, in particular the E1 region. When several genes ofinterest are used, they can be inserted at the same site or at differentsites in the viral genome and can be under the control of the sameregulatory elements or of independent elements and, where appropriate,some of them can be in the opposite orientation to the others in orderto minimize the phenomena of interference at the level of theirexpression. The genome of the recombinant adenoviral vector can beprepared by molecular biology techniques or by homologous recombination(see WO 96/17070).

The adenoviral vectors which are used within the context of the presentinvention are propagated in a complementing cell line which is able tosupply the defective function(s) in trans in order to produce thepeptides which are required for forming the infectious viral particles.For example, use will be made of cell line 293 for complementing the E1function (Graham et al., J. Gen. Virol., 1977, 36: 59-72) or of the celllines described in international application WO 97/04119 for effecting adouble complementation. It is also possible to employ an appropriatecell line and a helper virus in order to complement all the defectivefunctions. The viral particles which are produced are recovered from thecell culture and, if need be, purified using the techniques of the art(caesium chloride gradient, chromatographic steps, etc.).

The adenoviral vector which is used within the context of the presentinvention can be derived from the genome of an adenovirus of human,canine, avian, bovine, murine, ovine, porcine or simian origin or elsefrom a hybrid which comprises adenoviral genome fragments of differentorigins. Mention may be made, more specifically, of the CAV-1 or CAV-2adenoviruses of canine origin, of DAV of avian origin, or else of type 3Bad of bovine origin (Zakharchuk et al., Arch. Virol., 1993, 128:171-176; Spibey and Cavanagh, J. Gen. Virol., 1989,. 70: 165-172;Jouvenne et al., Gene, 1987, 60: 21-28; Mittal et al., J. Gen. Virol.,1995, 76: 93-102). However, preference will be given to an adenoviralvector that is specific for the particular animal species being studies.For example, porcine adenovirus (PAV) would be administered to pigs.

The method and means of the present invention may be embodied in theform of a kit.

The kit comprises a first container containing one or moreanti-inflammatory agents, a device for delivering the agents andinstructions for use.

In embodiments adapted -for use in intensive animal production, the kitmight additionally comprise a second container containing one or moreantibiotics. An alternate kit would comprise a first containercontaining one or more nucleic acid molecules encoding anti-inflammatoryagents, which when administered to an animal would, upon expression ofsaid nucleic acid molecule in the animal, produce a growth promotingamount of the anti-inflammatory agent, a device for delivering thenucleic acid molecules and instructions for use.

The instructions for use would enable a farmer or other animal husbandrypractitioner to administer the anti-inflammatory agent or nucleic acidmolecules such that growth promotion of the animal is enhanced relativeto an animal that is not administered such agents or nucleic acidmolecules.

Throughout the specification, the word “comprise” and variations of theword, such as “comprising” and “comprises”, means “including but notlimited to” and is not intended to exclude other additives, components,integers or steps.

The invention will now be further described by way of reference only tothe following non-limiting examples. It should be understood, however,that the examples following are illustrative only, and should not betaken in any way as a restriction on the generality of the inventiondescribed above. For example, while the majority of the examples relateto pigs, it is to be understood that the invention can also be appliedto other animals as disclosed herein, including for example, sheep,cattle and chickens.

EXAMPLE 1 Recombinant IL-1RA as a Growth Promotant to Replace In-FeedAntibiotics

This experiment was performed to determine whether IL-1ra is able toimprove the growth performance and health of weaner pigs in a commercialenvironment. We also wished to investigate the potential of IL-1ra as areplacement for antibiotic medication in feed.

Experiment Design

Recombinant porcine. IL-1ra was expressed in E. coli and purified usinga polyhis tag system. IL-1ra was tested for biological activity in abioassay prior to the start of the experiment.

Male weaner pigs (28 days old) were allocated to treatment groups of 20as described in Table 1. The mean weight for each treatment group wasequal, with equal variance. Pigs were housed in group pens of 20, with 2pens provided with medicated water and feed as per current industrystandards, while 2 pens were given unmedicated water and feed. Pigs wereinjected twice weekly with recombinant IL-1ra or saline (control), in avolume of 1 ml, for the duration of the weaner phase (42 days), asdescribed in Table 2.

Upon commencement of the weaner phase (day 0) and upon completion of theweaner phase (day 42), pigs were TABLE 1 Group treatment medicationSaline+ 1 ml saline Yes IL-1ra+ 200 μg IL-1ra Yes in 1 ml saline Saline−1 ml saline No IL-1ra− 200 μg IL-1ra No in 1 ml saline

TABLE 2 Day 0 Weighed and grouped 28 day old weaners. Bleed. InjectedGroups. Day 1 Injected Groups. Day 6 Weighed Injected Groups. Day 7(Week 1) Injected Groups. Day 9 Weighed Day 13 Injected Groups. InjectedGroups. Day 14 (Week 2) Weighed Day 16 Injected Groups. Day 20 InjectedGroups. Weighed Day 21 (Week 3) Injected Groups. Day 23 Injected Groups.Day 27 Weighed Injected Groups. Day 28 (Week 4) Injected Groups.Weighed. Final bleed. Day 30 Moved to grower pens. Day 34 Grower stage.All pigs given standard feed and remained in previous groups. Day 35(Week 5) Weighed during (D73) and end of grower Day 37 stage (D93). Day41 Finisher stage. Pigs moved into single pens and feed intake measuredfor FCR (food conversion ratio). All pigs given Day 42 (Week 6) standardfinisher feed. Weighed during (Days 42-93) (day of experiment (D) 114)and end of finisher stage (Slaughter D133). Measured final weight, P2backfat, (Days 93-133) carcass weight, % dressing.bled by venipuncture for immunological analyses. Pigs were weighedweekly throughout the weaner phase (day 0-day 42), and then on day 79,93 and at termination of the trial on day 113, after which, animals wereslaughtered and carcass characteristics measured.Results

For the first 4 weeks of treatment, pigs treated with IL-1ra performedas well as saline controls for rate of gain (FIG. 1). However, as theexperiment and treatments progressed into the later weeks of the weanerstage, weeks 5 and 6, pigs treated with IL-1ra were outperforming salinetreated pigs in both medicated and non-medicated feeding regimes (FIG.2). Furthermore, pigs treated with IL-1ra and fed a diet free ofantibiotics performed as well as saline treated pigs fed a medicateddiet (FIG. 2, IL-1ra—vs saline+). Pigs treated with IL-1ra and fed amedicated diet had the greatest rate of gain over the final 2 weeks ofweaning.

Such results suggest that IL-1ra treatment is as effective as in-feedantibiotic application for promoting the growth of pigs under commercialconditions. The improved performance of IL-1ra treated pigs after 4weeks of IL-1ra administration also implies that the effect of IL-1ra isdelayed, or requires several treatments to produce the similar effectsas in-feed antibiotics

The trend for higher rate of gain in IL-1ra treated pigs continuedthrough the grower and finisher phases (days 43-92, and days 93-134respectively). After injections ceased in week 6, pigs treated withIL-1ra without in-feed medication proceeded to grow as quickly as pigstreated with saline and provided with in-feed medication (FIG. 3).Again, this result implies that treatment with IL-1ra is as efficaciousas antibiotics for promoting increased growth in pigs, as shown in thelast 2 weeks of the weaner phase (FIG. 2) and in the grower phase (FIG.3, D79 and D93). The effect of IL-1ra appears to be long-lived after thecessation of cytokine treatment in week 6.

Pigs treated with IL-1ra and provided with a medicated diet showed thehighest rate of gain over the grower phase (FIG. 3, D79 and D93), whilepigs treated with saline and fed an unmedicated diet had the lowest rateof gain of all treatment groups.

In the finisher phase, pigs treated with IL-1ra during the weaner phasewithout a medicated diet showed the highest rate of gain of all groups(FIG. 4). This result further illustrates the delayed nature of responseto IL-1ra, which was administered until week 6, and that IL-1ra is aseffective a promoter of growth in pigs as the in-feed antibioticscurrently used by industry.

The positive effects of IL-1ra as a growth promotant are also indicatedby the average weight of pigs at slaughter (FIG. 5), and theirsubsequent warm carcass weight after slaughter (FIG. 6). Pigs treatedwith IL-1ra in the weaner phase in the absence of in-feed antibioticswere on average 0.7 kg heavier at slaughter than were pigs treated withsaline and provided with in-feed antibiotics. This increase in weightwith IL-1ra treatment occurred without any difference in feed conversionratio between these two treatments (FIG. 7). Pigs provided with in-feedantibiotics during the weaner phase had a feed conversion ratio of 2.5during the finisher phase, while pigs treated with IL-1ra in the absenceof in-feed antibiotics had a feed conversion ratio of 2.49 over thefinisher phase. The warm carcass weight of pigs treated with IL-1ra inthe presence of antibiotics was greater than that of theirsaline-treated counterparts (FIG. 6). Further, the warm carcass weightof pigs treated with IL-1ra in the absence of antibiotics was equal tothat obtained by pigs fed antibiotics and treated with saline. Theseresults illustrates that while IL-1ra treated pigs gained more weightthan antibiotic treated pigs, this gain was as efficient as the currentcommercial practice of feeding pigs antibiotics.

Conclusions

1). IL-1ra improved growth in pigs in the absence of in-feedantibiotics.

2). the improvement in growth produced by IL-1ra treatment was equal tothat seen by the addition of in-feed antibiotics.

3). IL-1ra improved growth in the last 2 weeks of the weaner phase inthe absence of in-feed antibiotics, compared to saline treated controls,and saline treated pigs fed an antibiotic supplemented diet.

4). The effect of IL-1ra administration during the weaner phase ongrowth was delayed and of long duration, continuing throughout thegrower and finisher phases.

5). IL-1ra treated pigs grew as well as antibiotic fed pigs in thegrower phase.

6). IL-1ra treated pigs grew faster than antibiotic fed pigs in thefinisher phase.

7). IL-1ra administration in the weaner phase resulted in increasedslaughter weights compared to pigs treated with saline and fed a dietsupplemented with antibiotics.

8). Pigs treated with IL-1ra in the weaner phase had the same feedefficiency during the finisher phase as pigs fed an antibioticsupplemented diet during the weaner phase.

9). These results indicate that IL-1ra produces larger pigs withoutaffecting feed conversion efficiency, than does the current industrypractice of supplementing pig diets with antibiotics.

EXAMPLE 2 Recombinant IL-1RA as a Growth Promotant to Reduce Levels ofIn-Feed Antibiotics

This experiment repeats the evaluation of IL-1ra to improve theperformance and/or immunity of pigs by comparing the growth rate andhealth of male and female weaner pigs through the weaner, grower andfinisher phases through to slaughter. This trial was designed toinvestigate the effect of providing IL-1ra at several levels ofmedication, from normal levels of antibiotic medication currently usedin pig production through to reduced antibiotic medication and absenceof antibiotic medication. This experiment evaluates the capacity ofIL-1ra to replace antibiotics under normal commercial pig rearingconditions, and determined the effect of continuous administration ofIL-1ra throughout the life of the animal.

Experiment Design

Recombinant porcine IL-1ra was expressed in E. coli and purified using apolyhis tag system. IL-1ra was tested for biological activity in abioassay prior to the start of the experiment.

The experiment was undertaken in a commercial environment where the pigswere weaned at 28 days of age. All injections were 1 ml. There were 16pigs per treatment, 8 males and 8 females per treatment. The mean weightof treatment groups were similar at the start of the experiment.

Treatment Protocol Group Treatment Medication 1 saline 0 2 salinereduced 3 saline normal 4 IL-1ra 0 5 IL-1ra reduced 6 IL-1ra normalSymbols Used

indicates no antibiotic supplements in feed or water throughout trial.

0.5 indicates single antibiotics used throughout trial.

+ indicates normal commercial antibiotic regime used throughout trial.

Treatments

A. Saline injection, 1 ml IM neck muscle.

B. 100 ug IL-1ra injection, 1 ml IM neck muscle. Injections wereadministered twice weekly during the weaner stage, and weekly throughoutthe grower and finisher stages.

Pigs were weaned and weighed at the commencement of the experiment (D0,W0). Weights were recorded weekly throughout the weaner phase (W0-W6),during the grower phase (W9), at the end of the grower phase (W13),during the finisher phase (W 16) and at the end of the finisher phaseprior to slaughter at W19. Blood samples were collected at the start ofthe experiment and at the end of the weaner phase, grower phase andfinisher phase. Haematology and immunological analyses were performed.At slaughter, carcass characteristics including P2 backfat measurementsand dressed carcass weight were noted.

Results

At the end of the weaner phase, pigs treated with IL-1ra weighed morethan their saline treated counterparts at all 3 levels of medication(FIG. 8). Significantly, the weight of weaner pigs treated with IL-1rawithout antibiotic medication was greater than the weight of weaner pigstreated with saline and provided normal levels of antibiotics (Irap− vsSaline+, FIG. 8). As expected, antibiotic medication enhanced the growthof saline treated pigs as well as improving the growth of pigs treatedwith IL-1ra.

The presence of antibiotic medication had a considerable effect on thehealth of pigs during the weaner phase as measured by the number of pigsexperiencing weight loss or mortality (FIG. 9). Increased levels ofantibiotics decreased the number of pigs experiencing weight loss ormortality in saline treated pigs. However, pigs treated with IL-1rashowed the least recordings of weight loss or mortality regardless ofantibiotic levels. In the absence of antibiotics, over half of the 16pigs treated with saline showed decreased health and production over theweaner period. However, this number was reduced to 2 out of 16 pigs withIL-1ra treatment (FIG. 9).

This improvement in health and decreased mortality over the weanerperiod, combined with improved weight gain resulted in markeddifferences for total group weights at the end of the weaner phase (FIG.10). Saline treated groups weighed 292 kg, 324 kg and 406 kg with nomedication, reduced medication and full medication respectively.Comparable total group weights at the end of the weaner phase for IL-1ratreated pigs were 369 kg, 390 kg and 451 kg. This increase in groupweight with IL-1ra treatment reflects an increase in productivity of26.4%, 20.4% and 11.1% at zero, reduced and normal levels of antibioticadministration respectively.

The improvement in productivity seen in the weaner phase with IL-1ratreatment was continued throughout the grower and finisher phases (FIGS.11 and 12). At the end of the grower phase, pigs treated with IL-1rawere heavier than all the saline treatment regardless of medicationlevel (FIG. 11). Importantly, delivery of IL-1ra in the absence ofantibiotics resulted in higher weights at the end of the grower phasethan the current industry level of medication. Finishing weight insaline treated pigs was affected by medication, in a dose-dependentmanner (FIG. 12), with increasing levels of medication resulting inincreased weight at slaughter. However, this pattern was not repeated inthe IL-1ra treated pigs. Pigs treated with IL-1ra weighed 16.7% morethan saline treated pigs in the absence of antibiotic medication.Indeed, IL-1ra administration without medication outperformed thecurrent industry level of antibiotic treatment in the promotion ofgrowth in finisher pigs (FIG. 12). These results suggest that IL-1ra ismore efficacious than antibiotic medication in promoting growth in pigsthroughout the production phases. Although IL-1ra treatment resulted inlarger pigs at slaughter, this increase in weight was not accompanied byan increase in P₂ backfat (FIG. 13). Pigs treated with IL-1ra in theabsence of antibiotics had P2 backfat levels that were comparable tothose of pigs treated with saline and provided full antibioticmedication (FIG. 13). Such results indicate that the improvement inliveweight seen with IL-1ra administration is carried through to the endproduct, resulting in leaner carcasses.

Conclusions

1). IL-1ra improved growth in pigs in the absence of in-feedantibiotics, and with reduced levels of antibiotics.

2). The improvement in growth produced by IL-1ra treatment exceeded thatseen by the current industry level of antibiotic medication.

3). IL-1ra improved growth throughout the production phases from weanerthrough to finisher, resulting in higher weights at slaughter.

4). IL-1ra improved the health of weaner pigs as seen by reducedmortalities and reduced incidence of weight losses compared with salinetreatment.

5). The improvement in health parameters seen with IL-1ra administrationwas greater than that provided by antibiotic medication.

6). Continuous administration of IL-1ra did not have any deleteriouseffects on pigs.

7). IL-1ra improved weight without compromising carcass quality as seenby unchanged P₂ backfat values at slaughter.

EXAMPLE 3 Delivery of Recombinant IL-1RA to Improve the Health of WeanerPigs—Infected with Haemorrhagic E. coli

This study determined whether IL-1ra was able to improve the health ofpigs exposed to infections, such as haemorrhagic E. coli. A further aimwas to determine whether IL-1ra could improve growth in pigs infectedwith E. coli at weaning. It was also designed to show whether IL-1racould reduce infection rates and improve health in pigs infected with E.coli. Finally it was hoped that the experiments would assess theprophylactic or therapeutic potential of IL-1ra for E. coli infectionsin weaner pigs compared to current antibiotic treatments.

Experiment Design

Male weaner pigs, with a mean weight of 5.4 kg were allocated to groupsof 8, with the mean weight being equalised between groups. Pigs werehoused in group pens containing a replicate from each treatment group.Pigs were provided with pelleted feed and water ad libitum.

Pigs were treated with recombinant saline, IL-1ra or the antibiotic,Apralan, and challenged with E. coli according to the schedule outlinedin Table 3. Saline, or 200 μg of IL-1ra were delivered intramuscularlyin 1 ml doses. Apralan was delivered orally according to manufacturer'sinstructions at a dose of 12 mg/kg. E. coli challenges were deliveredorally in an 8 ml dose containing 10⁸ cfu/ml. Blood was sampled frompigs by venipuncture at −2 days, day 0, and +6 days from initialchallenge with E. coli as outlined in Table 3. Blood was assayed forimmunological parameters including white blood cell number, differentialcell counts, lymphocyte subset enumeration, IgG levels and cytokineproduction. Pigs were weighed at day −2 and at the end of the trial onday 6.

Faecal samples were taken from each pig daily for 5 days from day 2 today 6 after challenge; these samples were cultured on sheep blood agarto quantify E. coli load. Growth on sheep blood agar was scored from 0to 5 (where 0 was no growth, 1 signified growth in the primary inoculum,2 signified growth in the first streak, 3 signified growth in the 2^(nd)streak, 4 signified growth in the 3^(rd) streak, and 5 signified growthof E. coli in the final streak). The condition of faeces was noted asnormal, wet or diarrhoea, as an indication of clinical signs.

At the conclusion of the experiment on day 7, pigs were euthanased andswabs were taken from different areas in the gastro-intestinal tract,including the small intestine (25%, 50% and 75% along the length of thesmall intestine), the caecum and colon, and from the faeces in situ.These post-mortem swabs were cultured on sheep blood agar to quantify E.coli load as described above. TABLE 3 EXPERIMENTAL PROTOCOL TO EXAMINETHE EFFICACY OF IL-1RA AS A PROPHYLACTIC FOR E. coli INFECTION IN PIGSDAY OF TRIAL EVENT −2 Blood sample IL-1ra injection Weights −1 IL-1rainjection Apralan orally 0 E. coli challenge IL-1ra injection Apralanorally Blood sample 1 E. coli challenge IL-1ra injection Apralan orally2 E. coli challenge IL-1ra injection Apralan orally Faecal swabs 3 E.coli challenge Apralan orally Faecal swabs 4 Faecal swabs 5 Faecal swabs6 Blood sample Faecal swabs Weights 7 Euthanase Swabs from gut andfaecesResults

Pigs treated with IL-1ra or Apralan showed decreased E. coli shedding infaeces compared to control pigs treated with saline (FIG. 14). Pigstreated with Apralan had reduced bacterial shedding from day. 2 to day 5after challenge, while IL-1ra treated pigs had reduced bacterialshedding from day 2 through to day 4. On day 6 after challenge,bacterial shedding from all groups was equal. For both Apralan andIL-1ra treatments, E. coli shedding in faeces returned to saline controllevels 3 days after the final treatment dose was delivered. Overall, theApralan treated group displayed the least bacterial shedding of alltreatments.

Faecal scores tallied over the entire challenge period for each groupshow an 80.9% decrease in faecal shedding for Apralan treated pigscompared to saline treated controls, while IL-1ra treated pigs showed a37% reduction in bacterial shedding compared to saline treated controls(FIG. 15).

In commercial situations, reduced bacterial shedding from infected pigswould further reduce cross-infection of other members of the herd orpen, thereby improving the health of weaners, and therefore growth.Enhancing the health and growth of weaner pigs would result in improvedproductivity in later phases since the major predictor of productivityis weight at the end of the weaner phase.

Clinical signs, recorded as the changes in faecal condition such as thepresence of wet faeces or diarrhoea, were decreased in pigs treated withIL-1ra or Apralan (FIG. 16). Pigs treated with IL-1ra had fewer recordedcases of wet faeces and diarrhoea than saline controls or Apralantreated pigs. Pigs treated with Apralan had fewer recordings of wetfaeces than did saline controls, but also displayed a minor increase inthe prevalence of diarrhoea in the post-challenge period (FIG. 16).

When these clinical signs are described as a percentage reduction insymptoms compared to saline controls, IL-1ra treatment produced a 64%reduction in clinical signs, while Apralan caused clinical symptoms tobe reduced by 27% (FIG. 17).

The results for clinical symptoms show that IL-1ra and Apralan were bothable to reduce the outward signs of infection with E. coli. In thismeasure of health, IL-1ra out-performed Apralan, the current antibiotictreatment for E. coli infections.

Both Apralan and IL-1ra treatments resulted in reduced bacterial load inall areas of the gastro-intestinal tract (GIT) compared withsaline-treated controls (FIG. 18). Pigs treated with IL-1ra recorded thelowest culture scores for all areas sampled in the small intestine andthe colon. IL-1ra and Apralan treated pigs had equally low faecalculture scores for samples taken from the caecum and faeces. IL-1ratreatment resulted in reductions of 71% E. coli in the anterior part ofthe small intestine, 51% reduction in the mid small intestine, 47% inthe posterior small intestine, 39% in the caecum, 44% in the colon and23% in faeces in situ compared to saline treated controls (FIG. 19).

When all culture scores were tallied for each pig and used to calculategroup mean total scores (FIG. 20), pigs treated with IL-1ra scored lessthan 10 out of a possible 30, compared with 17/30 for saline treatedpigs, and 12/30 for Apralan treated pigs. When this data is expressed asa percentage reduction in E. coli culture scores compared to salinecontrols (FIG. 21), prophylactic application of IL-1ra resulted in a 45%reduction in the amount of E. coli in the gastro-intestinal tract.Treatment with Apralan was only able to reduce E. coli colonisation inthe gut by 33%.

These results illustrate that bacterial load was lowest in pigs treatedwith IL-1ra, further emphasising the value of this preparation for thecontrol of haemorrhagic E. coli in young pigs.

When the post-mortem results for E. coli cultures were separated on thebasis of location in the gut, differences may be seen in the action ofIL-1ra and Apralan (FIG. 22). E. coli bacterial load in the smallintestine (foregut) correlates with the severity of disease, as thesmall intestine is the site in which the secretory diarrhoea ismanifested. Treatment with IL-1ra reduced the bacterial load in thesmall intestine by 55% compared with saline controls, while Apralancaused a reduction of 32% in the bacterial load in the small intestine(FIG. 23). In the hindgut area (caecum and colon), bacterial loadsrecorded for Apralan and IL-1ra treatments were similar, resulting in a37% and 42% reduction in E. coli respectively, compared to salinecontrols (FIG. 23).

The ability of IL-1ra to reduce bacterial load preferentially in theforegut would suggest that this treatment may reduce the severity ofdisease associated with haemorrhagic E. coli infection. Indeed, theseresults support those recorded for clinical signs of disease where pigstreated with IL-1ra had reduced incidence of diarrhoea compared to othertreatments (FIG. 16). Thus, IL-1ra may be a potential replacement oradjunct for the antibiotics currently administered in the pig industryto control the deleterious effects of this disease on pig production.

A summary of the comparative effects of IL-1ra and Apralan on bacterialshedding, clinical signs and bacterial load at post-mortem is includedin Table 4. TABLE 4 SUMMARY COMPARING THE THERAPEUTIC EFFECTS OF IRAP(IL-1RA) AND APRALAN FOR THE CONTROL OF HAEMORRHAGIC E. coli INFECTIONSIN YOUNG WEANER PIGS Change compared to saline treated controls ApralanIL-1ra Presence of bacterial ↓ 4 days ↓ 3 days shedding in faeces Changeon faecal ↓↓ 80.8% ↓ 36.5% bacterial load Change in clinical ↓ 27.3% ↓↓63.6% signs E. coli at post-mortem ↓ 32.9% ↓↓ 44.6% E. coli in foregut ↓32.1% ↓↓ 55.4% E. coli in hindgut ↓ 36.8% ↓ 41.7%Conclusions

1). IL-1ra improved the health of pigs i.e. it reduced the clinicalsigns of disease, in terms of faecal changes associated withhaemorrhagic diarrhoea in the presence of haemorrhagic E. coliinfection.

2). The improvement in health produced by IL-1ra treatment was equal to,and in some cases, greater than that produced by treatment with theantibiotic Apralan, the current method of treating haemorrhagic E. coliin pigs.

3). IL-1ra treatment resulted in decreased bacterial shedding in faecesduring the course of infection compared with saline-treated controls.Pigs treated with IL-1ra showed bacterial shedding significantly lessthan saline treated controls on 3/5 days after challenge. Such resultssuggest that under commercial conditions decreasing the bacterial loadin the environment may reduce infection rates.

4). The effect of IL-1ra administration resulted in decreased numbers ofbacteria in all areas of the GIT compared with saline treated controls.

5). Significantly, IL-1ra caused a 55% reduction in the bacterial loadin the small intestine (foregut), the site in which secretory diarrhoeais normally located during the course of E. coli infection. As bacterialload in the small intestine is associated with disease severity, IL-1ramay have a significant therapeutic effect on the progression andpathology of the disease.

6). IL-1ra treatment outperformed Apralan, the current antibiotictreatment used in industry, in reducing clinical signs of disease, E.coli levels present in the gut at post-mortem, in addition to E. colipresent in the crucial site of the small intestine.

EXAMPLE 4 Delivery of Recombinant IL-1RA to Improve the Health andProductivity of Weaner Pigs Infected with an Enteric InflammatoryPathogen Causing Swine Dysentery, Brachyspira (Serpulina) hyodysenteriae

The aim of this example was to determine whether IL-1ra could improvethe health of pigs infected with an enteric inflammatory pathogencausing swine dysentery, Brachyspira (Serpulina) hyodysenteriae. Afurther aim was to determine whether IL-4 could improve the growth rateof pigs under conditions of challenge with swine dysentery.

Experiment Design

Male pigs with a mean starting weight of 6.5 kg, were allocated totreatment groups consisting of eight pigs. Pigs were housed in grouppens, with each pen containing a replicate from each of the treatmentgroups. One group of 8 pigs was housed in a separate room and leftuninfected to act as untreated controls. Pigs were provided withpelleted feed and water ad libitum.

Prior to swine dysentery challenge, pigs were treated with 200 μgrecombinant IL-1ra or 1 ml saline via intramuscular injection. Lincocinwas delivered as a 2 ml intramuscular injection according to themanufacturer's instructions. Cytokines, antibiotics and challenges wereperformed at intervals described in the experimental protocol outlinedin Table 5. Pigs were infected with Brachyspira hyodysenteriae at day 0,day 1 and day 2, given as an oral bolus of 120 ml of spirochaete culturein log phase of growth, containing approximately 10⁸ cells.

Faecal swabs and blood samples were taken from each pig at intervalsdescribed in Table 5. Faecal swabs were cultured for the presence ofspirochaetes. Blood samples were assayed for immunological parameters asdescribed in Example 3 above. Pigs were weighed at weekly intervalsthroughout the experiment, which was terminated by euthanasia on days 19and 20 after the initial challenge. At post-mortem, swabs from areas ofthe hindgut were cultured for the presence of spirochaetes, and thegross pathological condition of the gastro-intestinal tissue noted.TABLE 5 PROTOCOL FOR EXPERIMENTAL PROCEDURES TO ASSESS THE EFFICACY OFIL-1RA AS A PROPHYLACTIC TREATMENT FOR SWINE DYSENTERY INFECTION Inject1 ml Inject Faecal IL-1ra, or 2 ml Day Weigh Swabs Infect Bleed SalineLincocin Kill −7 X −6 −5 −4 −3 −2 X −1 X 0 X X X X X 1 X X X 2 X X X 3 X4 5 X X X X 6 X 7 X X X X 8 X 9 X X X 10 11 12 X X X X 13 X 14 X X X X15 X 16 X X X 17 18 X X 19 X X 20 X X

Spirochaete cultures taken from the hindgut at post mortem show thattreatment of pigs with IL-1ra reduced the number of spirochaetesresiding in the gut compared to saline controls (FIG. 24). IL-1ra wasable to reduce spirochaete culture scores in the anterior colon,posterior colon and faeces compared to saline treated controls. Asexpected, pigs that were not challenged with swine dysentery did nothave spirochaetes in their hindgut or faeces at post mortem (data notshown).

Compared to saline treated pigs, IL-1ra treatment resulted in a 15.8%reduction in the anterior colon, 47.1% in the posterior colon and 42.1%reduction in faecal spirochaetes (FIG. 25). The net effect of IL-1ratreatment was a 27% reduction in spirochaetes throughout the GIT.

In addition to a reduction in the number of spirochaetes in the gut,treatment with IL-1ra also reduced the clinical signs associated withinfection indicated by faecal condition. FIG. 26 shows that IL-1ratreated pigs showed fewer signs of dysentery-affected faeces (wet andmucoid with blood) compared to saline treated controls.

Treatment with IL-1ra was able to reduce the production of thepro-inflammatory cytokines TNF, IL-8 and IL-1 (FIGS. 27, 28 and 29)compared to saline treated controls. Importantly, pro-inflammatorycytokines are associated with sickness behaviour in animals and havebeen implicated in reduced productivity seen in intensively housedlivestock. This anti-inflammatory ability of IL-1ra may translate tolong term improvements in growth. Indeed, such results have beendescribed for Examples 1, 2 and 3 above.

Furthermore, the clinical manifestation of swine dysentery is a chronicinflammatory pathology presumably exacerbated by inflammatory mediatorssuch as pro-inflammatory cytokines. The ability of IL-1ra to reduce theproduction of these inflammatory mediators may play a role in reducingthe pathology associated with swine dysentery infection.

Such results confirmed that IL-1ra was able to reduce the deleteriouseffect of swine dysentery infection on the health of pigs. IL-1ra wasknown to have anti-inflammatory effect on the immune system, thus, areduction in inflammatory pathological changes in the gut associatedwith dysentery may be attributable to both the anti-inflammatoryproperties of this cytokine and a reduced spirochaete load (as seen inFIG. 24).

Conclusions

1). Treatment of pigs with IL-1ra reduced the number of spirochaetespresent in the hindgut and faeces at post-mortem compared with salinetreatment.

2). IL-1ra reduced the clinical manifestation of swine dysenteryinfection as detected by faecal condition, compared with salinecontrols.

3). Treatment of pigs with IL-1ra resulted in reduced production ofpro-inflammatory cytokines which are associated with impaired growth andproductivity.

4). IL-1ra has been shown to improve the health of pigs in two entericinfection models: haemolytic E. coli and Brachyspira (Serpulina)hyodysenteriae (swine dysentery). Improvements in health in both modelswere described by reduced clinical symptoms during infection. In the E.coli model, the improvement in health was accompanied by a reduction ininfection associated pathology at post-mortem. In the swine dysenterymodel, reduced levels of pro-inflammatory cytokines were also noted. Theability of prophylactic treatment with IL-1ra to improve the health ofpigs exposed to E. coli was comparable to the performance of the currentindustry standards of antibiotic treatment. Thus, IL-1ra has potentialas an alternative, or supplement with, treatment to antibiotics, orpreventative, for E. coli and swine dysentery in pigs. The potential ofIL-1ra as a health promoter may be further enhanced by concurrentapplication with antibiotic therapeutics.

EXAMPLE 5 Delivery of Plasmids and Recombinant IL-1RA to Improve Growthand Health in Pigs Infected with Actinobacillus pleuropneumoniae

The aim of this example was to determine whether IL-1ra could improvethe health of pigs infected with the inflammatory lung pathogen,Actinobacillus pleuropneumoniae (App). Furthermore, another aim was todetermine whether IL-1ra could improve the growth rate of pigs underconditions of challenge with App. Additionally, this example aimed todetermine whether plasmid DNA or recombinant delivery of IL-1ra was moreefficacious.

Experiment Design

Male pigs, with a mean starting weight of 52 kg, were allocated to 5treatment groups as outlined in Table 6. Pigs were housed in group pens,with each pen containing a replicate from each of the treatment groups.The starting weights of each treatment group and each pen were equalprior to the start of the trial. Pigs were provided with pelleted feedand water ad libitum.

Recombinant IL-1ra and saline-were administered as 1 ml doses, givensubcutaneously behind the ear. Plasmids were administered in 1 ml doses,given intramuscularly in the hind-leg. Flunix was administered as a 2 mldose according to the manufacturer's instructions, and deliveredintramuscularly in the neck. The timetable of administration is outlinedin Table 6 below. TABLE 6 TREATMENTS AND DOSES APPLIED IN CYTOKINEEXPERIMENT (N = 4 PER GROUP) TO ASSESS THE EFFICACY OF IL-1RA AS APROPHYLACTIC TREATMENT ACTINOBACILLUS PLEUROPNEUMONIAE INFECTIONTREATMENT TREATMENT DOSE Saline 2 ml Flunix 2.2 mg/kg IL-1ra 100 μgPlasmid control 100 μg Plasmid IL-1ra 100 μg

Prior to challenge, pigs were treated with recombinant cytokines, flunixor plasmids as described Table 7. Pigs were anaesthetised and infectedintratracheally with 7.5×10⁵ pfu App on day 0.

Blood was sampled from pigs by venipuncture at 0, 24 h and 14 dayspost-challenge. Blood was assayed for immunological parameters aspreviously described. Pigs were weighed weekly from delivery of plasmidsand for 2 weeks after challenge. TABLE 7 PROTOCOL FOR EXPERIMENTALPROCEDURES TO ASSESS THE EFFICACY OF IL-4 AS A PROPHYLACTIC TREATMENTActinobacillus pleuropneumoniae INFECTION EVENT TIMING OF ADMINISTRATIONPlasmid delivery −10 days Recombinant delivery −2 days and day 0Challenge Day 0 Clinical visits 30 post-challengeResults

During the week of challenge, IL-1ra improved the growth of pigs (FIG.30) compared to saline-treated controls. Pigs treated with saline,flunix (a non-steroidal anti-inflammatory drug, NSAID), or controlplasmid showed weight loss, while pigs treated with IL-1ra or plasmidIL-1ra showed positive growth during the week of challenge. In the weekfollowing challenge, all groups of pigs gained weight, but again,recombinant IL-1ra treated pigs gained more weight on average than pigsin other treatment groups. Pigs treated with saline recoveredsignificantly in the second week after challenge, while pigs treatedwith IL-1ra continued to gain weight. Pigs treated with plasmids orflunix had the poorest growth of all groups in the second week ofchallenge.

Weight gain over the 2 week period following challenge with App (FIG.31) showed that recombinant IL-1ra treatment increased weight gaincompared to saline-treated controls, although this result was notstatistically significant. Flunix and plasmid-control were the poorestperforming treatments in terms of growth, compared to saline-treatedcontrols. IL-1ra plasmid performed better than the plasmid control groupin terms of growth over the 2 week period after challenge (FIG. 31).Similar patterns of performance were noted for daily rate of gain (FIG.32), with pigs treated with recombinant IL-1ra gaining on average 667 gper day compared to 433 g per day for saline treated control pigs.

Treatment with recombinant IL-1ra resulted in improvements in weightgain of 53.8% over saline controls, while treatment with the NSAIDflunix caused an 84.6% reduction in weight gain (FIG. 33). Plasmidtreatments generally had lower weight gain than did saline controlshowever, the IL-1ra plasmid improved weight gain by 100% compared to itsplasmid control (FIG. 34).

Pro-inflammatory cytokines, TNFα and IL-6 were elevated in severalgroups after challenge with App. Interestingly, the NSAID flunix, failedto inhibit the production of TNFα (FIG. 35), which may help to explainthe poor growth seen in this group. Recombinant IL-1ra, plasmid controland IL-1ra plasmid all had reduced levels of TNFα at day 13 afterchallenge compared with pre-challenge levels. These 3 treatments alsohad significantly lower levels of TNFα production than saline-treatedand flunix-treated pigs at day 13 after challenge (p<0.05).

All treatments reduced the production of IL-6 24 h after challengecompared with saline treated controls (FIG. 36), and this trendcontinued until 13d post-challenge. Unfortunately, IL-6 data was notretrievable for the saline treatment at 13 days after App challenge dueto sampling error. After 13 days of challenge, pigs treated with IL1-raas either plasmid or recombinant had reduced levels of thepro-inflammatory cytokine, IL-6, compared to pigs treated with flunix.

While the anti-inflammatory cytokine treatments did cause reductions inthe levels of pro-inflammatory cytokines in the circulation, and in somecases improved growth, the relationship between pro-inflammatorycytokines and impaired growth is still unclear. Generally, groups ofpigs with reduced levels of pro-inflammatory cytokines were the groupsthat also had the least inhibition of growth in the first week afterchallenge. Further work is required to elucidate the mechanism of weightloss in pigs under this challenge model.

In addition to improving the growth of pigs, we found that cytokinetreatment could improve the health of pigs exposed to App challenge. Thedata in FIG. 37 shows the mean clinical scores over 30 visits conductedduring the first week of challenge. The severity of symptoms displayedby each pig, such as lethargy, coughing and breathing parameters wasscored from 0-8, and pigs which died or were euthanased were arbitrarilygiven a score of 8 at each subsequent visit. Pigs treated withrecombinant IL-1ra had significantly reduced clinical signs of diseasecompared to saline-treated controls (p<0.05, FIG. 37). IL-1ra deliveredas a plasmid also resulted in reduced clinical symptoms compared tosaline and plasmid control pigs. Pigs treated with either saline orflunix showed the greatest clinical signs of App disease of alltreatment groups.

IL-1ra delivered as a recombinant caused a reduction of 72% in thepresence of clinical symptoms compared to pigs treated with saline (FIG.38). IL-1ra delivered in plasmid form produced a reduction of 52%compared to saline-treated controls, and 31% reduction compared toplasmid-treated controls (FIG. 38). IL-1ra delivered as plasmid orrecombinant was more effective than flunix in reducing the clinicalsymptoms of App infection.

At the conclusion of the trial, pigs were euthanased and the lungsremoved for post-mortem examination. Lungs were scored for pleurisy from0-5 (FIGS. 39 and 40) and the degree of pleuropneumonia was determinedby weighing affected lung and expressed as a percentage of total lungweight (FIGS. 41 and 42). Pigs treated with flunix and IL-1ra had lesspleurisy than the saline controls (FIG. 39). Although pigs treated withIL-1ra delivered as plasmid had less pleurisy than their plasmid-treatedcontrols, their level of pleurisy was comparable to that ofsaline-treated controls (FIG. 39). Recombinant IL-1ra reduced the levelsof pleurisy by 22% compared to saline treated controls, while treatmentwith flunix reduced pleurisy by 55.6% (FIG. 40). IL-1ra delivered asplasmid reduced pleurisy by 5.6% compared to saline treated controls,and 39.3% compared to plasmid controls (FIG. 40).

The percentage of lung affected by App lesions was greatly reduced inpigs treated with either flunix or recombinant IL-1ra compared withsaline-treated controls (FIG. 41). Both the saline control group, andthe plasmid control group had similar levels of lesion-affected lung.IL-1ra plasmid reduced the percentage of lung affected by App lesionswhen compared to saline and plasmid control groups, but the ability ofplasmid IL-1ra to impair the pathology of App disease is not aspronounced as IL-1ra delivered as a recombinant or flunix (FIG. 41).These results reflect a reduction in affected lung mass of 73.9% forIL-1ra, 64.1% for flunix and 36.7% for plasmid IL-1ra compared to salinetreated controls (FIG. 42).

Conclusions

1). Recombinant IL-1ra was able to greatly increase the growth of pigscompared to saline treated controls during the first week of Appchallenge. Pigs treated with IL-1ra were subsequently 4 kg heavier atthe termination of the experiment, after 2 weeks of challenge than theirsaline treated peers, which represents an improvement in growth of 69%.Pigs treated with flunix had the lowest growth over the 2 week challengeperiod.

2). Recombinant IL-1ra, plasmid control and plasmid IL-1ra were able toreduce the production of the pro-inflammatory cytokines TNFα and IL-6which are associated with poor growth performance. Flunix was able toreduce the production of IL-6 only.

3). IL-1ra greatly reduced the severity of clinical symptoms of diseaseduring the challenge, as did IL-1ra delivered as plasmid. RecombinantIL-1ra reduced clinical symptoms by 72%, while plasmid IL-1ra reducedclinical signs by 52% compared to saline treatment.

4). Flunix was able to reduce the level of pleurisy seen at post-mortem.IL-1ra reduced pleurisy by 22% compared to saline treatment.

5). Flunix, IL-1ra and plasmid IL-1ra all reduced the percentage of lungaffected by App lesions. Treatment

Blood was sampled from pigs by venipuncture at −24 h, +0 h, +24 h and +3weeks from challenge. Blood was assayed for immunological parameters aspreviously described. Pigs were weighed at day −1, day 10 and at 3weeks. TABLE 8 TREATMENTS AND DOSES APPLIED IN CYTOKINE EXPERIMENT (N =8 PER GROUP) TO ASSESS THE EFFICACY OF DIFFERENT DOSES OF IL-1RA ANDIL-1RA + IL-4 AS A PROPHYLACTIC TREATMENTS Actinobacilluspleuropneumoniae INFECTION TREATMENT TREATMENT DOSE saline 2 ml IL-1ralo 2 μg/kg IL-1ra hi 10/kg Synergy lo 2 μg/kg IL-4 + 2 μg/kg IL-1raSynergy hi 10 μg/kg IL-4 + 10 μg/kg IL-1raResults

Unlike Example 5, animals treated with saline did not experience weightloss during the early stages of challenge with App (compare FIG. 30 withFIG. 43). Despite this result, improvement in growth was seen in pigstreated with the high dose of IL-1ra (FIG. 43), equivalent to anincrease in rate of gain in excess of 100 g/day. Application of bothIL-1ra and IL-4 together to investigate synergy resulted in a depressedgrowth response during the first 10 days of App challenge compared tosaline treated pigs. Variation within groups was high, accounting forlarge error bars and lack of statistical significance in this instance.However, the trends of improved weight gain with IL-1ra seen in Example5 were repeated in this experiment.

In the last 10 days of challenge, pigs treated with low doses of IL-1raor low doses of IL1ra+IL-4 showed the greatest rate of gain (FIG. 44) at1250 g/day and 1306 g/day respectively, compared to 1079 g/day forsaline treated controls. Pigs treated with high doses of IL-1ra gained1170 g/day, which was higher than the rate of gain for saline treatment.The high synergy dose resulted in lower weight gain during the latterstages of App challenge.

Pigs treated with IL-1ra at low or high doses and low dose IL-1ra+IL-4exhibited higher weight gain than saline treated controls (FIG. 45).During the 21-day challenge period, pigs treated with low and high dosesof IL-1ra, or low dose synergy treatment gained 17.9 kg and 17 kgrespectively, while saline treated controls gained only 15.75 kg (FIG.45). Thus, treatment with IL-1ra or low dose IL-1ra+IL-4 improved growthby 13.5% and 7.9% respectively (FIG. 46) compared to saline treatedcontrols.

As seen in Example 5, treatment of pigs with recombinant IL-1ra causedreduced disease severity as recorded in pathology results at post-mortem(FIGS. 39, 40, 41 and 42). In the current example, application of IL-1raat the high dose reduced the amount of lung affected by App lesions(FIG. 47). Similarly, delivery of low dose IL-1ra and IL-4 combines alsoreduced affected lung weight compared to saline treatment. The degree ofpleurisy seen at post-mortem was reduced with high doses of IL-1ra andhigh doses of the synergy treatment (FIG. 48).

Production of the pro-inflammatory cytokine IL-8 was greatly reducedwith high dose treatment of IL-1ra and IL-1ra+IL-4 (FIG. 49) compared toother treatments. IL-8 recruits neutrophils to the lung and subsequentneutrophil degranulation is suspected to be a major factor in thepathology of App infection. Thus, reduction of IL-8 levels in lungtissue is likely to result in decreased pathology and improved health inpigs exposed to App infection. Similarly, the production of anotherpro-inflammatory cytokine, TNFα, was inhibited in lung tissue bytreatment with high doses of IL-1ra or low doses of IL-1ra+IL-4 (FIG.50). These results suggest that an anti-inflammatory mechanism may playa role in the beneficial effects of these treatments on the growth andhealth of pigs under conditions of App challenge.

Conclusions

1). IL-1ra at high doses improved growth early in challenge, whileIL-1ra at low or high doses, and low dose IL-1ra+IL-4 improved growth inthe latter stages of challenge.

2). IL-1ra at low or high doses, and low doses of IL-1ra+IL-4 resultedin increased weight gain over the entire challenge period.

3). IL-1ra at high doses and low dose IL-1ra+IL-4 reduced the amount oflung affected by App lesions. IL-1ra and IL-1ra+IL-4 at high dosesreduced pleurisy scores.

4). IL-1ra and IL-1ra+IL-4 at high doses had an anti-inflammatory effectas noted by reduced production of pro-inflammatory cytokines in lungtissue.

5). High doses of IL-1ra significantly decreased the production of IL-8in the lungs, which is associated with pathology

6). These results support the results of Example 5 which found animprovement in growth and a reduction in pathology with IL-1ra therapygiven prior to and at the time of infection with App.

EXAMPLE 7 Therapeutic Delivery of Recombinant IL-1RA at Low and HighDoses to Improve the Health and Growth of Pigs Infected withActinobacillus pleuropneumoniae

The aim of this example was to determine whether therapeutic delivery ofIL-1ra after Actinobacillus pleuropneumoniae (App) infection wasestablished, could abrogate infection and improve growth in pigs.

Experiment Design

Male pigs with a mean starting weight of 34.6 kg, were allocated to 4treatment groups of 9 pigs each. Treatments were saline, IL-1ra at 2 μgper kg, IL-1ra at 10 μg per kg and Excenel, the current clinicaltreatment for App infection. Pigs were housed in pens of 3 pigs, with 3replicates of each. Pigs were provided with pelleted feed and water adlibitum. The starting weights of each treatment group and each pen wereequal prior to the start of the trial.

Pigs were anaesthetised and infected intratracheally with 7.5×10⁵ pfu onday 0. Recombinant IL-1ra and saline were administered as 2ml doses,given subcutaneously behind the ear. Excenel was administered to pigsaccording to the manufacturer's instructions. Pigs were treated withIL-1ra, saline or Excenel at 24 h, 48 h and one week after challengewith App.

Blood was sampled from pigs by venipuncture at 0 h, 24 h, 48 h, 1 weekand 2 weeks after infection. Blood was assayed for immunologicalparameters as previously described. Pigs were weighed the day prior tochallenge, and days 6 and 13 after challenge.

Results

Mean weight gained in the second week after App challenge, illustratedin FIG. 51, shows that pigs treated with low doses of IL-1ra gained moreweight than other treatments. Pigs treated with low dose IL-1ra gainedon average 5.7 kg in the second week of challenge compared with 4.4 kgweight gain for saline treated controls, and 4.9 kg for antibiotictreatment (FIG. 51).

Furthermore, IL-1ra treatment reduced daily feed intake, as didtreatment with the antibiotic Excenel compared to saline treatment (FIG.52). Pigs treated with IL-1ra at low and high doses consumedrespectively 1.7 and 1.8 kg of feed per day, while pigs treated withExcenel consumed 2 kg and pigs treated with saline consumed 2.2 kg.

The combined effect of improved weight gain and decreased feed intakeresulted in improvements in feed conversion ratio (FCR, feed:gain) forpigs treated with low dose IL-1ra compared to saline controls (FIG. 53).IL-1ra treatment reduced FCR to 1.6 compared to 2.1 for salinetreatment.

Pigs treated with low dose IL-1ra tended to have improved proliferativeresponses of lymphocytes in the presence of killed App (FIG. 54).Lymphocyte proliferation assays measure the capacity of lymphocytes torespond to a particular antigen. In this case, the antigen washomologous with the infection and thus, high proliferative responses invitro are indicative of increased recognition of and mobilisationagainst the App pathogen in vivo. The trends seen for lymphocyteproliferative responses follow those for FCR—pigs that produced thegreatest in vitro response to killed App also had the greatest feedefficiency as evidenced by reduced FCR. Thus, IL-1ra may be improvingfeed efficiency and weight gain by enhancing specific immuneresponsiveness.

Therapeutic delivery of IL-1ra at low doses to pigs infected wit Appalso reduced the production of the pro-inflammatory cytokine IL-8 inlung tissue compared to other treatments (FIG. 55). Low dose IL-1ra andExcenel treatment also reduced the production of IL-8 in thecaudal-mediastinal lymph nodes, which drain the lungs, compared tosaline, treated controls (FIG. 56). Again, these results suggest thatIL-1ra is modulating protective immune responses and deleteriousinflammatory responses, which may contribute to improved weight gain andfeed conversion efficiency in pigs infected with App.

Conclusions

1). IL-1ra applied therapeutically at low doses improved weight gain inpigs infected with App, compared to antibiotic treatment or saline.

2). IL-1ra administered therapeutically decreased feed intake comparedto other treatments.

3). Therapeutic administration of IL-1ra greatly improved feedefficiency in pigs infected with App.

4). Therapeutic delivery of IL-1ra in pigs infected with App resulted inenhanced cellular immune responses while diminishing inflammatoryresponses.

1. A method for improving the growth performance of an animal comprisingthe step of administering to an animal in need thereof a growthpromoting amount of one or more anti-inflammatory agents.
 2. A methodaccording to claim 1, wherein the anti-inflammatory agent isadministered optionally in combination with a pharmaceutical carrier,adjuvant or vehicle.
 3. A method for improving the growth performance ofan animal comprising the step of administering to an animal in needthereof a composition comprising an anti-inflammatory agent inconjunction with an antibiotic, optionally in combination with apharmaceutical carrier, adjuvant or vehicle, wherein said compositionachieves a synergistic growth promoting effect.
 4. A method forimproving the growth performance of an animal comprising the step ofadministering to an animal in need thereof a compound or compositionwhich increases or supplements endogenous anti-inflammatory agentlevels, wherein growth performance is enhanced relative to the growthperformance of an animal which has not been administered said compoundor composition.
 5. A method according to claim 4, wherein the compoundor composition is administered prior to, together with, or subsequent tothe administration of a growth promoting amount of one or moreanti-inflammatory agents.
 6. A method according to claim 4 or claim 5,wherein the compound or composition comprises an antagonist of apro-inflammatory cytokine receptor.
 7. A method according to claim 6,wherein the antagonist is of TNF-α receptor, GM-CSF receptor, IL-6receptor, IL-1 receptor, IL-4 receptor or IL-8 receptor.
 8. A methodaccording to any one of claims 4 to 6, wherein the compound orcomposition comprises IL-10, 1,8-napthosultam substituted compounds orquinoxaline compounds.
 9. A method according to claim 4 or claim 5,wherein the compound or composition increases the endogenous level ofanti-inflammatory agents by decreasing the amount of pro-inflammatorycytokines.
 10. A method according to any one of claims 1 to 3 or 5,wherein the anti-inflammatory agent is a soluble cytokine receptor,cytokine receptor antagonist, cytokine inhibitory factor or biologicallyactive fragment thereof which has an anti-inflammatory effect or ananti-inflammatory agent selected from the group consisting ofdiclofenac, diflunisal, etodolac, flunix, fenoprofen, floctafenine,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate,mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin,phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic andtolmetin.
 11. A method according to claim 10, wherein the solublecytokine receptor or biologically active fragment thereof is selectedfrom the group consisting of TNFα receptor, IL-6 receptor, IL-1receptor, IL-4 receptor and IL-8 receptor or a combination thereof thatare capable of improving the growth performance of an animal.
 12. Amethod according to claim 10, wherein the soluble cytokine receptor orbiologically active fragment thereof is either IL-1 receptor, IL-4receptor, IL-8 receptor or a combination thereof.
 13. A method accordingto claim 10, wherein the soluble cytokine receptor or biologicallyactive fragment thereof is IL-1 receptor.
 14. A method according toclaim 10, wherein the cytokine receptor antagonist or biologicallyactive fragment thereof is selected from the group consisting of IL-1ra,IL-6ra, IL-8ra and TNF-αra.
 15. A method according to claim 14, whereinthe cytokine receptor antagonist or biologically active fragment thereofis IL-1ra.
 16. A method according to claim 10, wherein the cytokineinhibitory factor or biologically active fragment thereof is selectedfrom the group consisting of TNF blocking factor and TNF-alphainhibitor.
 17. A method according to any one of claims 1, 2, 4 to 16,further comprising the step of administering an antibiotic.
 18. A methodaccording to claim 3, wherein the step of administration the antibioticis prior to or subsequent to the administration of the anti-inflammatoryagent.
 19. A method according to claim 3 or claim 17, wherein theantibiotic is selected from the group consisting of amoxycylin,penicillin, procaine, ampicillin, cloxacillin, penicillin G, benzathine,benethamine, ceftiofur, cephalonium, cefuroxime, erythromycin, tylosin,tilmicosin, oleandomycin, kitasamycin, lincomycin, spectinomycin,tetracycline, oxytetracycline, chlortetracycline, neomycin, apramycin,streptomycin, avoparcin, dimetridazole, sulfonamides (includingtrimethoprim and diaveridine), bacitracin, virginiamycin, monensin,salinomycin, lasalocid, narasin and olaquindox or combinations thereof.20. A method according to claim 19, wherein the antibiotic is eitherlincomycin, spectinomycin or amoxicillin or combinations thereof.
 21. Amethod according to any one of claims 1 to 20, wherein theadministration is orally, topically, or parenterally.
 22. A methodaccording to claim 21, wherein parenteral administration is either bysubcutaneous injection, aerosol, intravenous, intramuscular,intrathecal, intrasternal injection, infusion techniques or encapsulatedcells.
 23. A method according to any one of claims 1 to 22, wherein theadministration is either a single dose unit or a multiple dose unit. 24.A method according to any one of claims 1 to 20, wherein theadministration is orally as an additive in water and/or feed.
 25. Amethod according to any one of claims 1 to 24, wherein the growthperformance of an animal is selected from the group consisting of anincrease in growth rate, an increase in efficiency of feed use, anincrease in final weight, an increase in dressed weight and decrease infat content.
 26. A method according to any one of claims 1 to 24,wherein the improved growth performance of an animal results fromimmunoenhancement, anti-parasitic or anti-microbial effects,anti-inflammatory effects or stress reduction.
 27. A method according toany one of claims 1 to 26, wherein the animal is either an Artiodactylor avian.
 28. A method according to claim 27, wherein the Artiodactyl isselected from the group consisting of cattle, pigs, sheep, camels, goatsand horses.
 29. A method according to claim 27, wherein the avian isselected from the group consisting of chickens, turkeys, geese and ducks30. A method according to claim 27, wherein the animal is cattle, pigs,or sheep.
 31. A growth promoting composition comprising one or moreanti-inflammatory agents together with one or more pharmaceuticalcarriers, adjuvants or vehicles.
 32. A growth promoting compositionaccording to claim 31, wherein the composition comprisesanti-inflammatory agents selected from the group consisting of solublecytokine receptor, cytokine receptor antagonist, cytokine inhibitingfactor or biologically active fragment thereof, diclofenac, diflunisal,etodolac, flunix, fenoprofen, floctafenine, flurbiprofen, ibuprofen,indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam,nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac,tenoxicam, tiaprofenic and tolmetin.
 33. A growth promoting compositionaccording to claim 32, wherein the composition comprises one or moresoluble cytokine receptor, cytokine receptor antagonist, cytokineinhibitory factor or biologically active fragments thereof and one ormore different soluble cytokine receptor, cytokine receptor antagonist,cytokine inhibitory factor or biologically active fragments thereof orone or more different anti-inflammatory agent.
 34. A growth promotingcomposition according to claim 32, wherein the composition comprises onesoluble cytokine receptor, cytokine receptor antagonist, cytokineinhibitory factor or biologically active fragment thereof and onedifferent anti-inflammatory agent or a pharmaceutical carrier, adjuvantor vehicle.
 35. A growth promoting composition according to claim 32,wherein the soluble cytokine receptor or biologically active fragmentthereof is selected from the group consisting of TNFα receptor, IL-6receptor, IL-1 receptor, IL-4 receptor and IL-8 receptor or acombination thereof.
 36. A growth promoting composition according toclaim 32, wherein the soluble cytokine receptor or biologically activefragment thereof is either IL-1 receptor, IL-4 receptor, IL-8 receptoror a combination thereof.
 37. A growth promoting composition accordingto claim 32, wherein the soluble cytokine receptor or biologicallyactive fragment thereof is IL-1 receptor.
 38. A growth promotingcomposition according to claim 32, wherein the cytokine receptorantagonist or biologically active fragment thereof is selected from thegroup consisting of IL-1ra, IL-6ra, IL-8ra and TNF-αra.
 39. A growthpromoting composition according to claim 32, wherein the cytokinereceptor antagonist or biologically active fragment thereof is IL-1ra.40. A growth promoting composition according to claim 32, wherein thecytokine inhibitory factor or biologically active fragment thereof isselected from the group consisting of TNF blocking factor and TNF-alphainhibitor.
 41. A growth promoting composition according to any one ofclaims 31 to 40, further comprising one or more antibiotics.
 42. Agrowth promoting composition according to claim 41, wherein theantibiotic is selected from the group consisting of amoxycylin,penicillin, procaine, ampicillin, cloxacillin, penicillin G, benzathine,benethamine, ceftiofur, cephalonium, cefuroxime, erythromycin, tylosin,tilmicosin, oleandomycin, kitasamycin, lincomycin, spectinornycin,tetracycline, oxytetracycline, chlortetracycline, neomycin, apramycin,streptomycin, avoparcin, dimetridazole, sulfonamides (includingtrimethoprim and diaveridine), bacitracin, virginiamycin, monensin,salinomycin, lasalocid, narasin and olaquindox or combinations thereof.43. A growth promoting composition according to claim 42, wherein theantibiotic is lincomycin, spectinomycin or amoxicillin or combinationsthereof.
 44. A method for improving the growth performance of an animalcomprising the step of administering to an animal in need thereof anucleic acid molecule encoding one or more anti-inflammatory agents,wherein the expression of said nucleic acid molecule produces aneffective growth promoting amount of one or more anti-inflammatoryagents.
 45. A method according to claim 44, wherein the nucleic acidmolecule is DNA, cDNA, RNA, or a hybrid molecule thereof.
 46. A methodaccording to claim 44 or claim 45, wherein the nucleic acid molecule isa full-length molecule or a biologically active fragment thereof.
 47. Amethod according to any one of claims 30 to 32, wherein the nucleic acidmolecule is a DNA molecule encoding a soluble cytokine receptor,cytokine receptor antagonist, cytokine inhibitory factor or biologicallyactive fragment thereof.
 48. A method according to 47, wherein the DNAencodes a cytokine receptor selected from the group consisting of TNFαreceptor, IL-6 receptor, IL-1 receptor, IL-4 receptor and IL-8 receptoror a combination thereof, or a cytokine receptor antagonist selectedfrom the group consisting of IL-1ra, IL-6ra and TNF-αra.
 49. A methodaccording to any one of claims 44 to 48, wherein the nucleic acidmolecule either integrates into the animal genome or is anextrachromosomal element.
 50. A method according to any one of claims 44to 49, wherein the nucleic acid molecule is administered by injectionsubcutaneously, intravenously, or intramuscularly or administered as anaerosol.
 51. A method according to claim 50, wherein the nucleic acidmolecule is administered in an amount of about 1 μg to 2000 μg per dose52. A method according to claim 50, wherein the nucleic acid molecule isadministered in an amount of about 5 μg to 1000 μg per dose.
 53. Amethod according to claim 52, wherein the nucleic acid molecule isadministered in an amount of about 6 μg to 200 μg per dose.
 54. A methodaccording to any one of claims 44 to 53, wherein the nucleic acidmolecule is administered in a vector or as naked DNA.
 55. A methodaccording to claim 54, wherein the vector is a porcine adenovirusvector.
 56. A construct for delivering in vivo an effective amount of acytokine receptor, cytokine receptor antagonist, cytokine inhibitoryfactor or biologically active fragment thereof, comprising: a) anucleotide sequence encoding a cytokine receptor, cytokine receptorantagonist, cytokine inhibitory factor or biologically active fragmentthereof; b) a vector comprising a control sequence wherein the controlsequence is capable of the controlling the expression of the nucleotidesequence of a) such that a cytokine receptor, cytokine receptorantagonist, cytokine inhibitory factor or biologically active fragmentthereof is produced which in turns improves growth performance in ananimal.
 57. A kit used for improving the growth performance of an animalcomprising: a). one or more anti-inflammatory agents; b). a deliverydevice for said anti-inflammatory agents; and c). instructions for usein the method of the invention.